Modulators of aldehyde dehydrogenase activity and methods of use thereof

ABSTRACT

The present invention provides compounds that function as modulators of aldehyde dehydrogenase activity; and pharmaceutical compositions comprising the compounds. The present invention provides therapeutic methods involving administering a subject compound, or a subject pharmaceutical composition.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.13/717,056, filed Dec. 17, 2012, now U.S. Pat. No. 8,906,942 which is adivisional of U.S. application Ser. No. 12/553,805 filed Sep. 3, 2009,now U.S. Pat. No. 8,354,435, which claims the benefit of U.S.Provisional Patent Application No. 61/095,227, filed Sep. 8, 2008, thedisclosures of each of which applications are incorporated herein byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under contract AA011147awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

Aldehyde dehydrogenases (ALDH) constitute a family of enzymes that playa critical role in detoxification of various cytotoxic xenogenic andbiogenic aldehydes. There are at least 19 members/isozymes of the ALDHfamily, where the various isozymes may exhibit different substratespecificity and/or cellular location relative to other members of thefamily.

Cytotoxic aldehydes derive from a variety of sources. For example,environmental (external) sources of aldehydes include those that resultfrom ethanol consumption, consumption of food sources, or from ingestionof hazardous materials such as vinyl chloride, pesticides, herbicides,etc. Aldehydes that may be cytotoxic can also be produced biologically,e.g., as a result of oxidative stress such as occurs in ischemia,irradiation, or metabolism or bioconversion of cellular precursors suchas neurotransmitters and drugs. Accumulation of cytotoxic levels ofaldehydes, and/or defects in the ALDH enzyme, has been implicated in avariety of diseases and conditions, or in increased risk of diseasedevelopment. The range of implicated diseases includes neurodegenerativediseases, aging, cancer, myocardial infarction, stroke, dermatitis,diabetes, and liver diseases.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) is encoded in the nucleargenome and is transported into mitochondria. ALDH2 is a tetramericprotein composed of four identical subunits, each consisting of 500amino acid residues. This tetramer can be regarded as a dimer of dimers.The interface between monomers that form a dimer is different and moreextensive than the interface between the two dimers that form thetetramer. Each subunit is composed of three main domains: the catalyticdomain, the coenzyme or NAD⁺-binding domain, and the oligomerizationdomain.

There is a need in the art for modulators of aldehyde dehydrogenaseenzymatic activity.

LITERATURE

-   Larson et al. (2005) J. Biol. Chem. 280:30550; Li et al. (2006) J.    Clin. Invest. 116:506; US Patent Publication No. 2005/0171043; WO    2005/057213; WO 2008/112164.

SUMMARY OF THE INVENTION

The present invention provides compounds that function as modulators ofaldehyde dehydrogenase activity; and pharmaceutical compositionscomprising the compounds. The present invention provides therapeuticmethods involving administering a subject compound, or a subjectpharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B provide the amino acid sequence of human ALDH2 (SEQID NO:1) and the amino acid sequence of an E487K variant of human ALDH2,respectively.

FIGS. 2A and 2B provide exemplary ALDH1 amino acid sequences.

FIG. 3 provides an exemplary ALDH3 amino acid sequence.

FIG. 4 schematically depicts a fluorescent aldehyde dehydrogenaseenzymatic assay.

DEFINITIONS

As used herein, the term “aldehyde dehydrogenase” or “ALDH” refers to anenzyme that oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenicaldehyde, or an aldehyde produced from a compound that is ingested,inhaled, or absorbed) to its corresponding acid in an NAD⁺-dependent oran NADP⁺-dependent reaction. For example, ALDH oxidizes aldehydesderived from the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, or that are producedduring normal metabolism. An example of a biogenic aldehyde isacetaldehyde produced as a product of alcohol dehydrogenase activity oningested ethanol.

As used herein, the term “aldehyde dehydrogenase” or “ALDH” refers to anenzyme that oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenicaldehyde, or an aldehyde produced from a compound that is ingested,inhaled, or absorbed) to its corresponding acid in an NAD⁺-dependent oran NADP⁺-dependent reaction. For example, ALDH oxidizes aldehydesderived from the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, that are produced as aresult of oxidative stress, or that are produced during normalmetabolism, e.g., conversion of retinaldehyde to retinoic acid. Anexample of a biogenic aldehyde is acetaldehyde produced as a product ofalcohol dehydrogenase activity on ingested ethanol. An aldehydedehydrogenase can also exhibit esterase activity and/or reductaseactivity.

The term “ALDH” encompasses ALDH found in the cytosol, in themitochondria, microsome, or other cellular compartment. The term “ALDH”encompasses ALDH found primarily in one or a few tissues, e.g., cornea,saliva, liver, etc., or in stem cells and embryos. The term “ALDH”encompasses any of the known ALDH isozymes, including ALDH1, ALDH2,ALDH3, ALDH4, ALDH5, etc.

As used herein, the term “mitochondrial aldehyde dehydrogenase-2” or“ALDH2” refers to an enzyme that oxidizes an aldehyde (e.g., a xenogenicaldehyde, a biogenic aldehyde, or an aldehyde produced from a compoundthat is ingested, inhaled, or absorbed) to its corresponding acid in anNAD⁺-dependent reaction. For example, ALDH2 oxidizes aldehydes derivedfrom the breakdown of compounds, e.g., toxic compounds that areingested, that are absorbed, that are inhaled, or that are producedduring normal metabolism. Mitochondrial ALDH2 is naturally found inmitochondria.

The term “ALDH2” encompasses ALDH2 from various species. Amino acidsequences of ALDH2 from various species are publicly available. Forexample, a human ALDH2 amino acid sequence is found under GenBankAccession Nos. AAH02967 and NP_000681; a mouse ALDH2 amino acid sequenceis found under GenBank Accession No. NP_033786; and a rat ALDH2 aminoacid sequence is found under GenBank Accession No. NP_115792. The term“ALDH2” encompasses an aldehyde dehydrogenase that exhibits substratespecificity, e.g., that preferentially oxidizes aliphatic aldehydes. Theterm “ALDH2” encompasses an enzymatically active polypeptide having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 18-517 of the aminoacid sequence set forth in SEQ ID NO:1 (FIG. 1A) or SEQ ID NO:2 (FIG.1B).

The term “ALDH2” as used herein also encompasses fragments, fusionproteins, and variants (e.g., variants having one or more amino acidsubstitutions, addition, deletions, and/or insertions) that retain ALDH2enzymatic activity. Specific enzymatically active ALDH2 variants,fragments, fusion proteins, and the like can be verified by adapting themethods described herein. An example of an ALDH2 variant is an ALDH2polypeptide that comprises a Glu-to-Lys substitution at amino acidposition 487 of human ALDH2, as depicted in FIG. 1B (amino acid 504 ofSEQ ID NO:2), or at a position corresponding to amino acid 487 of humanALDH2. This mutation is referred to as the “E487K mutation”; the “E487Kvariant”; or as the “Glu504Lys polymorphism”. See, e.g., Larson et al.(2005) J. Biol. Chem. 280:30550; and Li et al. (2006) J. Clin. Invest.116:506. An ALDH2 variant retains at least about 1% of the enzymaticactivity of a corresponding wild-type ALDH2 enzyme. For example, theE487K variant retains at least about 1% of the activity of an enzymecomprising the amino acid sequence depicted in FIG. 1A (SEQ ID NO:1).“ALDH2” includes an enzyme that converts acetaldehyde into acetic acid,e.g., where the acetaldehyde is formed in vivo by the action of alcoholdehydrogenase on ingested ethanol.

As used herein, “ALDH1” refers to a cytosolic aldehyde dehydrogenasethat oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenicaldehyde, or an aldehyde produced from a compound that is ingested,inhaled, or absorbed) to its corresponding acid in an NAD⁺-dependentreaction.

The term “ALDH1” encompasses ALDH1 from various species. Amino acidsequences of ALDH1 from various species are publicly available. See,e.g., GenBank Accession Nos. AAC51652 (Homo sapiens ALDH1); NP_000680(Homo sapiens ALDH1); AAH61526 (Rattus norvegicus ALDH1); AAI05194 (Bostaurus ALDH1); and NP_036051 (Mus musculus ALDH1). The term “ALDH1” asused herein also encompasses fragments, fusion proteins, and variants(e.g., variants having one or more amino acid substitutions, addition,deletions, and/or insertions) that retain ALDH1 enzymatic activity. Theterm “ALDH1” encompasses an aldehyde dehydrogenase that oxidizesaromatic aldehydes, including those of the naphthaldehyde,phenanthrenealdehyde, and coumarinaldehyde series, as well as complexpolyaromatic aldehydes. The term “ALDH1” encompasses a cytosolicaldehyde dehydrogenase. ALDH1 does not accept the coenzyme NADP⁺, butinstead uses the coenzyme NAD⁺.

The term “ALDH1” encompasses an enzymatically active polypeptide havingat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:3 or SEQ ID NO:4 (depicted in FIGS. 2A and 2B,respectively).

The term “ALDH3” encompasses ALDH3 from various species. Amino acidsequences of ALDH3 from various species are publicly available. See,e.g., GenBank Accession Nos. AAB26658 (Homo sapiens ALDH3), NP_000683(Homo sapiens ALDH3), P30838 (Homo sapiens ALDH3), NP_001106196 (Musmusculus ALDH3), and AAH70924 (Rattus norvegicus ALDH3). The term“ALDH3” as used herein also encompasses fragments, fusion proteins, andvariants (e.g., variants having one or more amino acid substitutions,addition, deletions, and/or insertions) that retain ALDH3 enzymaticactivity. The term “ALDH3” encompasses an aldehyde dehydrogenase thatexhibits specificity toward aromatic aldehydes, e.g., oxidizing aromaticaldehydes of the 2-naphthaldehyde series, but inactive toward1-naphthaldehydes and higher polyaromatic aldehydes. The term “ALDH3”encompasses an aldehyde dehydrogenase that can use both NAD⁺ and NADP⁺as co-substrate. The term “ALDH3” encompasses aldehyde dehydrogenasefound naturally in saliva and in the cornea.

The term “ALDH3” encompasses an enzymatically active polypeptide havingat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:5 (as depicted in FIG. 3).

The term “ALDH5” (also referred to as “succinic semialdehydedehydrogenase”) encompasses an NAD⁺-dependent enzyme that oxidizessuccinic semialdehyde to succinate. ALDH5 is involved in the catabolismof 4-aminobutyric acid (GABA). Naturally-occurring ALDH5 can be found inthe mitochondria of eukaryotic cells. The term “ALDH5” encompasses anenzymatically active polypeptide having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in GenBank Accession No.AAH34321.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature. Isolated compounds are at least about80%, at least about 90% pure, at least about 95% pure, at least about98% pure, or at least about 99% pure, by weight. The present inventionis meant to comprehend diastereomers as well as their racemic andresolved, enantiomerically pure forms and pharmaceutically acceptablesalts thereof.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient, in combination withanother agent, or alone in one or more doses, to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to a member or members of any mammalian ornon-mammalian species that may have a need for the pharmaceuticalmethods, compositions and treatments described herein. Subjects andpatients thus include, without limitation, primate (including humans),canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)),avian, and other subjects. Humans and non-human mammals havingcommercial importance (e.g., livestock and domesticated animals) are ofparticular interest.

“Mammal” refers to a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, e.g., humans. Non-human animal models,particularly mammals, e.g. a non-human primate, a murine (e.g., a mouse,a rat), lagomorpha, etc. may be used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectcompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for the novel unit dosage formsof the present invention depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes one and morethan one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and is free of contaminants that are capable of eliciting anundesirable response within the subject (e.g., the compound(s) in thepharmaceutical composition is pharmaceutical grade). Pharmaceuticalcompositions can be designed for administration to subjects or patientsin need thereof via a number of different routes of administrationincluding oral, buccal, rectal, parenteral, intraperitoneal,intradermal, intracheal and the like. In some embodiments thecomposition is suitable for administration by a transdermal route, usinga penetration enhancer other than dimethylsulfoxide (DMSO). In otherembodiments, the pharmaceutical compositions are suitable foradministration by a route other than transdermal administration. Apharmaceutical composition will in some embodiments include a subjectcompound and a pharmaceutically acceptable excipient. In someembodiments, a pharmaceutically acceptable excipient is other than DMSO.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and are either pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

A “pharmaceutically acceptable ester” of a compound of the inventionmeans an ester that is pharmaceutically acceptable and that possessesthe desired pharmacological activity of the parent compound, andincludes, but is not limited to, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl estersof acidic groups, including, but not limited to, carboxylic acids,phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids andboronic acids.

A “pharmaceutically acceptable enol ether” of a compound of theinvention means an enol ether that is pharmaceutically acceptable andthat possesses the desired pharmacological activity of the parentcompound, and includes, but is not limited to, derivatives of formulaC═C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

“Pro-drugs” means any compound that releases an active parent drugaccording to one or more of the generic formulas shown below in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of one or more of the generic formulas shown below are preparedby modifying functional groups present in the compound of the genericformula in such a way that the modifications may be cleaved in vivo torelease the parent compound. Prodrugs include compounds of one or moreof the generic formulas shown below wherein a hydroxy, amino, orsulfhydryl group in one or more of the generic formulas shown below isbonded to any group that may be cleaved in vivo to regenerate the freehydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to esters (e.g., acetate, formate, andbenzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) ofhydroxy functional groups in compounds of one or more of the genericformulas shown below, and the like.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combinations thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Halogens of particular interest include chloro and bromogroups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature, or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

A subject compound may possess one or more asymmetric centers; suchcompounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anALDH agonist” includes a plurality of such agonists and reference to“the pharmaceutical composition” includes reference to one or morepharmaceutical compositions and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides compounds that function as modulators ofaldehyde dehydrogenase (ALDH) enzymatic activity, as well ascompositions and formulations comprising the compounds. The presentinvention provides therapeutic methods involving administering a subjectcompound, or a subject pharmaceutical composition.

Agonists of ALDH (e.g., ALDH1, ALDH2, ALDH3, ALDH4, ALDH5, etc.) areuseful for treating a variety of disorders, including, e.g., conditionsinvolving ischemic stress, chronic free-radical associated diseases,acute free-radical associated diseases, insensitivity to nitroglycerin(e.g., in angina and heart failure), hypertension, diabetes, andosteoporosis. Agonists of ALDH are also useful for reducing the level inan individual of a compound such as ethanol, methanol, ethylene glycolmonomethyl ether, polyvinyl chloride, xenogenic aldehydes, and biogenicaldehydes. Agonists of ALDH are also useful for reducing the level in anindividual of a compound that, when ingested, absorbed, or inhaled,gives rise to an aldehyde substrate for ALDH. The present inventionprovides therapeutic methods involving administering a subject compound,or a subject pharmaceutical composition.

In some embodiments, individuals to be treated are humans. In someembodiments, a human to be treated according to a subject method is onethat has two “wild-type” ALDH2 alleles, e.g., the ALDH2 encoded by thetwo wild-type ALDH2 alleles has a glutamic acid at position 487, asdepicted in FIG. 1A. In other embodiments, a human to be treatedaccording to a subject method is one that has one or two “ALDH2*2”alleles, e.g., the ALDH2 encoded by one or both ALDH2 alleles comprisesa lysine as amino acid position 487, as depicted in FIG. 1B. The E487Kpolymorphism is a semidominant polymorphism, and results in an ALDH2tetramer that has significantly lower enzymatic activity than“wild-type” ALDH2. Thus, individuals who are heterozygous or homozygousfor the ALDH2*2 allele have much lower in vivo ALDH2 activity levelsthan individuals who are homozygous for the “wild-type” ALDH2 allele.Individuals who are heterozygous or homozygous for the ALDH2*2 alleleare expected to benefit from treatment with a subject ALDH2 agonist,because the level of ALDH2 activity in such individuals is particularlylow, and any increase of ALDH2 activity levels would be expected toprovide a therapeutic effect. Any increase in ALDH2 activity would bebeneficial in treating conditions such as ischemic disorders, inincreasing the responsiveness of such individuals to nitroglycerin,etc., as discussed in more detail below.

Modulators of Aldehyde Dehydrogenase

The present invention provides compounds that function as modulators ofaldehyde dehydrogenase (ALDH) activity; and pharmaceutical compositionscomprising the compounds. The present invention provides compounds thatfunction as modulators of mitochondrial aldehyde dehydrogenase-2 (ALDH2)activity; and pharmaceutical compositions comprising the compounds.

In some embodiments, a subject ALDH agonist increases activity of a“wild-type ALDH2 enzyme. In other embodiments, a subject ALDH agonistincreases activity of a variant ALDH2 enzyme that comprises an E→Ksubstitution at amino acid 487 of the mature enzyme. In someembodiments, a human to be treated according to a subject method is onethat has two “wild-type” ALDH2 alleles, e.g., the ALDH2 encoded by thetwo wild-type ALDH2 alleles has a glutamic acid at position 487 of themature protein (amino acid 504 of the protein including the leaderpeptide), as depicted in FIG. 1A. In other embodiments, a human to betreated according to a subject method is one that has one or two“ALDH2*2” alleles, e.g., the ALDH2 encoded by one or both ALDH2 allelescomprises a lysine as amino acid position 487 of the mature protein(amino acid 504 of the protein including the leader peptide), asdepicted in FIG. 1B. The E487K polymorphism is a semidominantpolymorphism, and results in an ALDH2 tetramer that has significantlylower enzymatic activity than “wild-type” ALDH2. Thus, individuals whoare heterozygous or homozygous for the ALDH2*2 allele have much lower invivo ALDH2 activity levels than individuals who are homozygous for the“wild-type” ALDH2 allele. Individuals who are heterozygous or homozygousfor the ALDH2*2 allele are expected to benefit from treatment with asubject ALDH2 agonist, because the level of ALDH2 activity in suchindividuals is particularly low, and any increase of ALDH2 activitylevels would be expected to provide a therapeutic effect. Any increasein ALDH2 activity would be beneficial in treating conditions such asischemic disorders, in increasing the responsiveness of such individualsto nitroglycerin, etc., as discussed in more detail below.

In some embodiments, a subject ALDH agonist selectively modulates (e.g.,increases) an enzymatic activity of a particular ALDH isozyme. Forexample, in some embodiments, a subject ALDH agonist selectivelyincreases an enzymatic activity of ALDH1. For example, in someembodiments, a subject ALDH agonist increases an enzymatic activity ofALDH1, but does not substantially increase the same enzymatic activityof an ALDH isozyme other than ALDH1, e.g., the ALDH agonist increases anenzymatic activity of an ALDH isozyme other than ALDH1, if at all, by nomore than about 15%, e.g., by less than 15%, less than 10%, less than5%, or less than 1%.

In some embodiments, a subject ALDH agonist selectively modulates (e.g.,increases) an enzymatic activity of a particular ALDH isozyme. Forexample, in some embodiments, a subject ALDH agonist selectivelyincreases an enzymatic activity of ALDH2. For example, in someembodiments, a subject ALDH agonist increases an enzymatic activity ofALDH2, but does not substantially increase the same enzymatic activityof an ALDH isozyme other than ALDH2, e.g., the ALDH agonist increases anenzymatic activity of an ALDH isozyme other than ALDH2, if at all, by nomore than about 15%, e.g., by less than 15%, less than 10%, less than5%, or less than 1%.

In some embodiments, a subject ALDH agonist selectively modulates (e.g.,increases) an enzymatic activity of a particular ALDH isozyme. Forexample, in some embodiments, a subject ALDH agonist selectivelyincreases an enzymatic activity of ALDH3. For example, in someembodiments, a subject ALDH agonist increases an enzymatic activity ofALDH3, but does not substantially increase the same enzymatic activityof an ALDH isozyme other than ALDH3, e.g., the ALDH agonist increases anenzymatic activity of an ALDH isozyme other than ALDH3, if at all, by nomore than about 15%, e.g., by less than 15%, less than 10%, less than5%, or less than 1%.

In some embodiments, a subject ALDH agonist increases enzymatic activityof both ALDH2 and ALDH1. In some embodiments, a subject ALDH agonistincreases enzymatic activity of both ALDH2 and ALDH1, but does notsubstantially increase enzymatic activity of an ALDH isozyme other thanALDH2 and ALDH1. In some embodiments, a subject ALDH agonist increasesenzymatic activity of both ALDH1 and ALDH2, where the ALDH2 comprises alysine at amino acid 487 of the mature protein (amino acid 504 of theprotein including the leader peptide) as depicted in FIG. 1B.

A subject ALDH agonist will in some embodiments increases an enzymaticactivity of an ALDH for a particular substrate or class of substrates.For example, in some embodiments, a subject ALDH agonist increases anenzymatic activity of an ALDH3 enzyme for complex polyaromatic aldehydessuch as phenanthrenealdehyde. As another example, a subject ALDH agonistincreases an enzymatic activity of an ALDH1 enzyme for a substrate suchas phenylacetaldehyde. As another example, a subject ALDH agonistincreases an enzymatic activity of an ALDH1 enzyme for a naphthaldehydederivative of the phenanthrene series. As another example, a subjectALDH agonist increases an enzymatic activity of an ALDH3 enzyme for along-chain aliphatic aldehyde (e.g., 6-methyoxy-2-naphthaldehyde;2-naphthaldehyde; 6-dimethylamino-2-naphthaldehyde; etc.). As anotherexample, a subject ALDH agonist increases an enzymatic activity of anALDH2 enzyme (either wild-type or E487K variant, or both) foracetaldehyde.

In some embodiments, a compound that modulates ALDH activity modulates adehydrogenase activity of ALDH, e.g., the compound modulatesdehydrogenase activity in oxidizing an aldehyde (e.g., a xenogenicaldehyde, a biogenic aldehyde, or an aldehyde produced from a compoundthat is ingested, inhaled, or absorbed) to the corresponding acid. Inother embodiments, a compound that modulates ALDH activity modulates anesterase activity of ALDH. In other embodiments, a compound thatmodulates ALDH activity modulates a reductase activity of ALDH. Forexample, ALDH can convert nitroglycerin to nitric oxide (NO) via itsreductase activity.

As noted above, in some embodiments, a compound that modulates ALDHactivity modulates a dehydrogenase activity of ALDH, e.g., the compoundmodulates dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid.

A variety of compounds can give rise to aldehyde substrates for ALDH(e.g., ALDH2). Non-limiting examples of compounds that can give rise toaldehyde substrates for ALDH include ethanol; a variety of insecticides;industrial toxins such as vinyl chlorides (e.g., polyvinyl chloride);and pyruvate. For example, a compound is ingested, absorbed (e.g.,through the skin), or inhaled, by a mammal and is subsequently convertedin the mammal into an aldehyde substrate for ALDH.

Biogenic aldehydes include aldehydes that are produced by a mammal,e.g., are produced metabolically by a mammal. Non-limiting examples ofbiogenic aldehydes include ω-6 polyunsaturated fatty acids, such asmalondialdehyde (MDA); 3,4-dihydroxypheylacetaldehyde (DOPAL);3,4-dihydroxyphenylglycolaldehye (DOPEGAL); hexanal; acrolein; glyoxal;crotonaldehyde; trans-2-nonenal; 4-oxo-2-nonenal; and4-hydroxy-2-nonenal (HNE) (see e.g., Ellis, Pharmacology & Therapeutics(2007) 115:13, Picklo and Montine (2007) J Alzheimers Dis. 12:185);3-aminopropanal (3-AP), a product of polyamine oxidase; and aldehydeproducts of tyrosine, serine and threonine (see Wood et al, Brain Res(2006) 1095; 190); and retinaldehdye (see e.g. Chen et al, MolecularPharmacology (1994) 46:88). A further example of a biogenic aldehyde isacetaldehyde formed as a product of alcohol dehydrogenase activity oningested ethanol.

Xenogenic aldehydes include aldehydes ingested, absorbed, or inhaled bya mammal from source outside the mammal. Xenogenic aldehydes include,e.g., formaldehyde and glutaraldehyde (e.g., McGregor et al., Crit RevToxicol (2006) 36:821 and Pandey et al Hum Exp. Toxicol. (2000) 19:360);chloroacetaldehyde (see e.g., Richardson et al., Mutat. Research (2007)636:178); and reactive aldehydes present in cigarette smoke (see Simthet al., Inhal. Toxicol. (2006) 18:667).

Non-limiting examples of compounds that are substrates for ALDH include3,4-dihydroxypheylacetaldehyde (DOPAL); 3,4-dihydroxyphenylglycolaldehye(DOPEGAL); acrolein; formaldehyde; acetaldehyde; propionaldehyde;n-butyraldehyde; capronaldehyde; heptaldehyde; pentaldehyde;octylaldehyde; decylaldehyd; retinaldehyde; 3-hydroxybenzaldehyde;2,5-dihydroxybenzaldehyde; phenylacetaldehyde; 3-phenylpropionaldehyde(see, e.g., Want et al. (2002) Drug Metabolism and Disposition 30:69);cinnamoyl and hydrocinnamoyl aldehydes and their derivative aldehydes(e.g. p-nitrocinnamaldehyde, p-(dimethylamino)cinnamaldehyde,hydrocinnamaldehyde, phenylpropionaldehyde); benzaldehyde and itsderivative aldehydes (e.g. 2,4-dinitro-benzaldehyde,o-nitro-benzaldehyde, p-nitro-benzaldehyde, p-methyl-benzaldehyde,m-methyl-benzaldehyde, p-methoxy-benzaldehyde,p-(dimethylamino)-benzaldehyde, m-methoxy-benzaldehyde,m-hydroxy-benzaldehyde, 3,4-dimethoxy-benzaldehyde,o-methoxy-benzaldehyde); naphthaldehyde and its derivative aldehydes(e.g. 5-bromo-1-naphthaldehyde, 5-nitro-1-naphthaldehyde,6-[O—(CH₂)₅—COOH]-2-naphthaldehyde, 6-(dimethylamino)-2-naphthaldehyde);coumarin-4-carboxaldehyde and its derivative aldehydes (e.g.7-acetoxy-coumarin-4-carboxaldehyde,7-(dimethylamino)-coumarin-4-carboxaldehyde,7-methoxy-coumarin-4-carboxaldehyde,6,7-dimethoxy-coumarin-4-carboxaldehyde); quinoline,quinolinonecarboxaldehyde, and their derivative aldehydes (e.g.quinoline-3-carboxaldehyde,7-(dimethylamino)-2-quinolinone-4-carboxaldehyde,quinoline-4-carboxaldehyde, 6-methoxy-2-quinolinone-4-carboxaldehyde);phenanthrene-9-carboxaldehyde; indole-3-aldehyde, indole-3-actaldehyde;5-methoxyindole-3-carboxaldehyde; 3-pyridinecarboxaldehyde;fluorene-2-carboxaldehyde (see, e.g., Klyosov, (1996) Biochemistry35:4457); 4-hydroxynonenal; malondialdehyde;3,4-dihydroxyphenylacetaldehyde; and 5-hydroxylindole-3-acetaldehyde.See, also, e.g., Williams et al. (2005) Anal. Chem. 77:3383; Marchittiet al. (2007) Pharmacol. Rev. 59:125; and Hoffman and Maser (2007) DrugMetab. Rev. 39:87.

ALDH Agonists

The present invention provides ALDH agonists (also referred to as“activators”); and pharmaceutical compositions comprising ALDH agonists.In other embodiments, a subject ALDH agonist is selective for ALDH2. Insome embodiments, a subject ALDH agonist is also an agonist for one ormore ALDH isozymes in addition to ALDH2. In some embodiments, a subjectALDH agonist is also an agonist for ALDH2 and ALDH1. In otherembodiments, a subject ALDH agonist is also an agonist for ALDH2 andALDH3. In some embodiments, a one or more of the compounds discussed inWO 2008/112164 is specifically excluded.

A subject ALDH agonist is useful for treating a variety of disorders,including, e.g., conditions involving ischemic stress, chronicfree-radical associated diseases, acute free-radical associateddiseases, insensitivity to nitroglycerin (e.g., in angina and heartfailure), hypertension, diabetes, seizures, cancer, acute myocardialinfarction, stroke, skin damage, dermatitis, atherosclerosis,Alzheimer's Disease, Parkinson's Disease, and osteoporosis. A subjectALDH agonist is also useful in the detoxification of alcohol abuse,methanol poisoning, ethylene glycol monomethyl ether poisoning, andpoisoning due to other xenogenic or biogenic aldehyde compounds.

In some embodiments, a subject ALDH agonist exhibits enhanced solubilityin a physiological solution. A “physiological solution” includes variousbodily fluids (e.g., blood, serum, plasma, cerebrospinal fluid, urine,etc.); buffers such as a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-morpholino)ethanesulfonic acid (MES),2-(N-morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS); aphysiological saline solution (e.g., 0.9% NaCl); and the like. In someembodiments, a subject ALDH agonist has a solubility of from about 10 mMto about 100 mM or more (e.g., from about 10 mM to about 25 mM, fromabout 25 mM 50 mM, from about 50 mM to about 75 mM, from about 75 mM toabout 100 mM, or greater than 100 mM) in 0.9% NaCl at a temperature inthe range of from about 22° C. to about 40° C. (e.g., from about 22° C.to about 25° C., from about 25° C. to about 30° C., from about 30° C. toabout 35° C., or from about 35° C. to about 40° C.).

In some embodiments, a subject ALDH agonist exhibits enhanced solubilityin a physiological solution, compared to a compound described in, e.g.,WO 2008/112164. For example, in some embodiments, a subject ALDH agonistexhibits at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 50%, at least about75%, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 5-fold, at least about 7-fold, or at least about10-fold, or greater than 10-fold, increased solubility in a liquid(e.g., a biologically compatible liquid; a pharmaceutically acceptableliquid), compared to the solubility of another ALDH agonist (e.g., acompound described in WO 2008/112164) in the same liquid. As an example,in some embodiments, a subject ALDH agonist has a solubility of fromabout 10 mg/ml to about 1 g/ml in water at 22° C., e.g., in someembodiments, a subject ALDH agonist has a solubility of from about 10mg/ml to about 25 mg/ml, from about 25 mg/ml to about 50 mg/ml, fromabout 50 mg/ml to about 100 mg/ml, from about 100 mg/ml to about 200mg/ml, from about 200 mg/ml to about 300 mg/ml, from about 300 mg/ml toabout 400 mg/ml, from about 400 mg/ml to about 500 mg/ml, from about 500mg/ml to about 600 mg/ml, from about 600 mg/ml to about 700 mg/ml, fromabout 700 mg/ml to about 800 mg/ml, or from about 800 mg/ml to about 1g/ml in water at 22° C. In some embodiments, a subject ALDH agonist hasa solubility of from about 10 mg/ml to about 25 mg/ml, from about 25mg/ml to about 50 mg/ml, from about 50 mg/ml to about 100 mg/ml, fromabout 100 mg/ml to about 200 mg/ml, from about 200 mg/ml to about 300mg/ml, from about 300 mg/ml to about 400 mg/ml, from about 400 mg/ml toabout 500 mg/ml, from about 500 mg/ml to about 600 mg/ml, from about 600mg/ml to about 700 mg/ml, from about 700 mg/ml to about 800 mg/ml, orfrom about 800 mg/ml to about 1 g/ml in an aqueous solution at 22° C.solubility of from about 10 mg/ml to about 25 mg/ml, from about 25 mg/mlto about 50 mg/ml, from about 50 mg/ml to about 100 mg/ml, from about100 mg/ml to about 200 mg/ml, from about 200 mg/ml to about 300 mg/ml,from about 300 mg/ml to about 400 mg/ml, from about 400 mg/ml to about500 mg/ml, from about 500 mg/ml to about 600 mg/ml, from about 600 mg/mlto about 700 mg/ml, from about 700 mg/ml to about 800 mg/ml, or fromabout 800 mg/ml to about 1 g/ml in an organic solvent at 22° C. In someembodiments, a subject ALDH agonist has a solubility of from about 1mg/ml to about 10 mg/ml in water at 22° C., in an aqueous solution at22° C., or in an organic solvent at 22° C.

In some embodiments, a subject ALDH agonist exhibits enhancedbioavailability, e.g., increased oral bioavailability. For example, thebioavailability can be greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90%, greater than 95%, greater than 98%, or greater than 99%.

A subject ALDH agonist increases an enzymatic activity of an ALDHpolypeptide by at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100% (or two-fold), at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 20-fold, at least about 25-fold, or at leastabout 50-fold, or greater than 50-fold, when compared to the enzymaticactivity of the ALDH polypeptide in the absence of the agonist.

In some embodiments, a subject ALDH agonist increases a dehydrogenaseactivity (e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH polypeptide by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases the esteraseactivity of an ALDH polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100% (or two-fold),at least about 2.5-fold, at least about 5-fold, at least about 10-fold,at least about 15-fold, at least about 20-fold, at least about 25-fold,or at least about 50-fold, or greater than 50-fold, when compared to theesterase activity of the ALDH polypeptide in the absence of the agonist.

In some embodiments, a subject ALDH agonist increases the reductaseactivity of an ALDH polypeptide by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 100% (or two-fold),at least about 2.5-fold, at least about 5-fold, at least about 10-fold,at least about 15-fold, at least about 20-fold, at least about 25-fold,or at least about 50-fold, or greater than 50-fold, when compared to thereductase activity of the ALDH polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an enzymaticactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to theenzymatic activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases a dehydrogenaseactivity (e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an esteraseactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to theesterase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases a reductaseactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:1 (depicted in FIG. 1A), or as set forth in aminoacids 18-517 of SEQ ID NO:1, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thereductase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an enzymaticactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forth in aminoacids 18-517 of SEQ ID NO:2, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to theenzymatic activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases a dehydrogenaseactivity (e.g., dehydrogenase activity in oxidizing an aldehyde (e.g., axenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from acompound that is ingested, inhaled, or absorbed) to the correspondingacid) of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forth in aminoacids 18-517 of SEQ ID NO:2, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thedehydrogenase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an esteraseactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forth in aminoacids 18-517 of SEQ ID NO:2, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to theesterase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases a reductaseactivity of an ALDH2 polypeptide comprising an amino acid sequence setforth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forth in aminoacids 18-517 of SEQ ID NO:2, by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100% (or two-fold), atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, orat least about 50-fold, or greater than 50-fold, when compared to thereductase activity of the ALDH2 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an enzymaticactivity (e.g., an aldehyde dehydrogenase activity, a reductaseactivity, or an esterase activity) of an ALDH1 polypeptide comprising anamino acid sequence set forth in SEQ ID NO:3 or 4 (depicted in FIGS. 2Aand 2B, respectively), by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 100% (or two-fold), at leastabout 2.5-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, or atleast about 50-fold, or greater than 50-fold, when compared to theenzymatic activity of the ALDH1 polypeptide in the absence of theagonist.

In some embodiments, a subject ALDH agonist increases an enzymaticactivity (e.g., an aldehyde dehydrogenase activity, a reductaseactivity, or an esterase activity) of an ALDH3 polypeptide comprising anamino acid sequence set forth in SEQ ID NO:5 (depicted in FIG. 3), by atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 100% (or two-fold), at least about 2.5-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 50-fold, or greaterthan 50-fold, when compared to the enzymatic activity of the ALDH3polypeptide in the absence of the agonist.

In some embodiments, a subject ALDH agonist is specific for (e.g.,selective for) ALDH2, e.g., a subject ALDH2 agonist increases anenzymatic activity of an ALDH2 enzyme, but does not substantiallyincrease the same enzymatic activity of cytosolic aldehydedehydrogenase-1 (ALDH1), e.g., a subject ALDH2 agonist increases anenzymatic activity of an ALDH1 enzyme, if at all, by less than about15%, less than about 10%, less than about 5%, less than about 2%, orless than about 1%, when used at a concentration that increases the sameenzymatic activity of an ALDH2 enzyme by at least about 5% or more. Insome embodiments, a subject ALDH2 agonist does not substantiallyincrease the enzymatic activity of alcohol dehydrogenase (ADH), e.g., asubject ALDH2 agonist increases the enzymatic activity of an ADH, if atall, by less than about 5%, less than about 2%, or less than about 1%,when used at a concentration that increases the enzymatic activity of anALDH2 enzyme by at least about 5% or more.

For example, in some embodiments, a subject ALDH agonist is specific for(e.g., selective for) ALDH2, e.g., a subject ALDH2 agonist increasesdehydrogenase activity of an ALDH2 enzyme, but does not substantiallyincrease the dehydrogenase activity of cytosolic aldehydedehydrogenase-1 (ALDH1), e.g., a subject ALDH2 agonist increasesdehydrogenase activity of an ALDH1 enzyme, if at all, by less than about15%, less than about 10%, less than about 5%, less than about 2%, orless than about 1%, when used at a concentration that increasesdehydrogenase activity of an ALDH2 enzyme by at least about 5% or more.In some embodiments, a subject ALDH2 agonist does not substantiallyincrease dehydrogenase activity of alcohol dehydrogenase (ADH), e.g., asubject ALDH2 agonist increases the dehydrogenase activity of an ADH, ifat all, by less than about 5%, less than about 2%, or less than about1%, when used at a concentration that increases the dehydrogenaseactivity of an ALDH2 enzyme by at least about 5% or more.

In some embodiments, a subject ALDH agonist is specific for (e.g.,selective for) ALDH1, e.g., a subject ALDH agonist increases anenzymatic activity of an ALDH1 enzyme, but does not substantiallyincrease the same enzymatic activity of any other ALDH isozyme, e.g., asubject ALDH agonist increases an enzymatic activity of an ALDH isozymeother than ALDH1, if at all, by less than about 15%, less than about10%, less than about 5%, less than about 2%, or less than about 1%, whenused at a concentration that increases the same enzymatic activity of anALDH1 enzyme by at least about 15% or more.

In some embodiments, a subject ALDH agonist is specific for (e.g.,selective for) ALDH3, e.g., a subject ALDH agonist increases anenzymatic activity of an ALDH3 enzyme, but does not substantiallyincrease the same enzymatic activity of any other ALDH isozyme, e.g., asubject ALDH agonist increases an enzymatic activity of an ALDH isozymeother than ALDH3, if at all, by less than about 15%, less than about10%, less than about 5%, less than about 2%, or less than about 1%, whenused at a concentration that increases the same enzymatic activity of anALDH3 enzyme by at least about 15% or more.

In some embodiments, a subject ALDH agonist increases an enzymaticactivity of both ALDH1 and ALDH2, but does not does not substantiallyincrease the same enzymatic activity of any other ALDH isozyme, e.g., asubject ALDH agonist increases an enzymatic activity of an ALDH isozymeother than ALDH1 and ALDH2, if at all, by less than about 15%, less thanabout 10%, less than about 5%, less than about 2%, or less than about1%, when used at a concentration that increases the same enzymaticactivity of an ALDH1 and ALDH2 enzyme by at least about 15% or more.

In some embodiments, a subject ALDH agonist has an EC₅₀ of from about 1nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nMto about 15 nM, from about 15 nM to about 25 nM, from about 25 nM toabout 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about100 nM, from about 100 nM to about 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 450 nM, from about 450 nM to about500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, fromabout 75 μM to about 100 μM, from about 100 μM to about 250 μM, fromabout 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

For example, in some embodiments, a subject ALDH agonist has an EC₅₀ offrom about 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM,from about 10 nM to about 15 nM, from about 15 nM to about 25 nM, fromabout 25 nM to about 50 nM, from about 50 nM to about 75 nM, from about75 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 450 nM, from about 450 nM toabout 500 nM, from about 500 nM to about 750 nM, from about 750 nM toabout 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,from about 75 μM to about 100 μM, from about 100 μM to about 250 μM,from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM,for a dehydrogenase activity of mitochondrial ALDH2.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ for an ALDH2polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1(depicted in FIG. 1A), or as set forth in amino acids 18-517 of SEQ IDNO:1, of from about 1 nM to about 1 mM, e.g., from about 1 nM to about10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM,from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, fromabout 75 nM to about 100 nM, from about 100 nM to about 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 450 nM, fromabout 450 nM to about 500 nM, from about 500 nM to about 750 nM, fromabout 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, from about 75 μM to about 100 μM, from about 100 μM toabout 250 μM, from about 250 μM to about 500 μM, or from about 500 μM toabout 1 mM.

For example, in some embodiments, a subject ALDH2 agonist has an EC₅₀for dehydrogenase activity of an ALDH2 polypeptide comprising an aminoacid sequence set forth in SEQ ID NO:1 (depicted in FIG. 1A), or as setforth in amino acids 18-517 of SEQ ID NO:1, of from about 1 nM to about1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15nM, from about 15 nM to about 25 nM, from about 25 nM to about 50 nM,from about 50 nM to about 75 nM, from about 75 nM to about 100 nM, fromabout 100 nM to about 150 nM, from about 150 nM to about 200 nM, fromabout 200 nM to about 250 nM, from about 250 nM to about 300 nM, fromabout 300 nM to about 350 nM, from about 350 nM to about 400 nM, fromabout 400 nM to about 450 nM, from about 450 nM to about 500 nM, fromabout 500 nM to about 750 nM, from about 750 nM to about 1 μM, fromabout 1 μM to about 10 μM, from about 10 μM to about 25 μM, from about25 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μMto about 100 μM, from about 100 μM to about 250 μM, from about 250 μM toabout 500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ for an ALDH2polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2(depicted in FIG. 1B), or as set forth in amino acids 18-517 of SEQ IDNO:2, of from about 1 nM to about 1 mM, e.g., from about 1 nM to about10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM,from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, fromabout 75 nM to about 100 nM, from about 100 nM to about 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 450 nM, fromabout 450 nM to about 500 nM, from about 500 nM to about 750 nM, fromabout 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, from about 75 μM to about 100 μM, from about 100 μM toabout 250 μM, from about 250 μM to about 500 μM, or from about 500 μM toabout 1 mM.

In some embodiments, a subject ALDH2 agonist has an EC₅₀ fordehydrogenase activity of an ALDH2 polypeptide comprising an amino acidsequence set forth in SEQ ID NO:2 (depicted in FIG. 1B), or as set forthin amino acids 18-517 of SEQ ID NO:2, of from about 1 nM to about 1 mM,e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15 nM,from about 15 nM to about 25 nM, from about 25 nM to about 50 nM, fromabout 50 nM to about 75 nM, from about 75 nM to about 100 nM, from about100 nM to about 150 nM, from about 150 nM to about 200 nM, from about200 nM to about 250 nM, from about 250 nM to about 300 nM, from about300 nM to about 350 nM, from about 350 nM to about 400 nM, from about400 nM to about 450 nM, from about 450 nM to about 500 nM, from about500 nM to about 750 nM, from about 750 nM to about 1 μM, from about 1 μMto about 10 μM, from about 10 μM to about 25 μM, from about 25 μM toabout 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about100 μM, from about 100 μM to about 250 μM, from about 250 μM to about500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH agonist has an EC₅₀ for an enzymaticactivity (e.g., an aldehyde dehydrogenase activity, an esteraseactivity, a reductase activity) of an ALDH1 polypeptide of from about 1nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nMto about 15 nM, from about 15 nM to about 25 nM, from about 25 nM toabout 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about100 nM, from about 100 nM to about 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 450 nM, from about 450 nM to about500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, fromabout 75 μM to about 100 μM, from about 100 μM to about 250 μM, fromabout 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH agonist has an EC₅₀ for an enzymaticactivity (e.g., an aldehyde dehydrogenase activity, an esteraseactivity, a reductase activity) of an ALDH3 polypeptide of from about 1nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nMto about 15 nM, from about 15 nM to about 25 nM, from about 25 nM toabout 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about100 nM, from about 100 nM to about 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 450 nM, from about 450 nM to about500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, fromabout 75 μM to about 100 μM, from about 100 μM to about 250 μM, fromabout 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

In some embodiments, a subject ALDH2 agonist is a compound of genericFormula I, as shown below:

where each of R₁, R₂, and R₃ is independently selected from H; a halo(e.g., bromo, fluoro, chloro, iodo); a substituted or unsubstitutedphenyl group; an aliphatic group, an alkyl group; a substituted alkylgroup; an alkenyl group; an alkynyl group; a substituted orunsubstituted cyclic group; a substituted or unsubstituted heterocyclicgroup; a substituted or unsubstituted aryl group; and a substituted orunsubstituted heteroaryl group;

where A is C or S and where a=1 when A=C; and where a=2 when A=S; and

where Ar₁ and Ar₂ are independently selected from a substituted arylgroup, an unsubstituted aryl group, a substituted heteroaryl group, andan unsubstituted heteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

For example, in some embodiments, Ar₁ of Formula I are independently:

where R₄-R₈ are each independently selected from H; a halo (e.g., bromo,fluoro, chloro, iodo); a substituted or unsubstituted phenyl group; analiphatic group, an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; asubstituted or unsubstituted heterocyclic group; a substituted orunsubstituted aryl group; and a substituted or unsubstituted heteroarylgroup. In other embodiments, Ar₁ of Formula I are independently asubstituted or unsubstituted heterocyclic group, e.g., a substituted orunsubstituted pyridine, a thiazole, an imidazole, a thiophene, aquinoline, an isoquinoline, or a furan group.

In exemplary embodiments of Formula I, Ar₂ of Formula I is independentlya substituted pyridine oxide. For example, in exemplary embodiments ofFormula I, Ar₂ of Formula I is independently a substituted pyridineoxide selected from the formulas,

where R₉ to R₁₂ is each independently selected from H; a halo (e.g.,bromo, fluoro, chloro, iodo); an aliphatic group; an alkyl group; asubstituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; an ether; a substituted orunsubstituted amine; an ester; and an amide group.

In some embodiments, a subject ALDH agonist is a compound of genericFormula II, as shown below:

where X_(n) and X_(y) are each independently H, C, N, O, or a halogen(e.g., F, Br, Cl, or I); where n is the integer 0 or 1; where y is theinteger 0 or 1;

where . . . (dotted line) is an optional bond; where z is the integer 0,1, or 2;

where A is C or S, and where a=1 when A=C; and where a=2 when A=S;

where Ar is an unsubstituted or substituted aryl group, a substitutedheteroaryl group, or an unsubstituted heteroaryl group; and

where R₁ to R₆ is each independently selected from H; a halo (e.g.,bromo, fluoro, chloro, iodo); a substituted or unsubstituted phenylgroup; an aliphatic group, an alkyl group; a substituted alkyl group; analkenyl group; an alkynyl group; a substituted or unsubstituted cyclicgroup; a substituted or unsubstituted heterocyclic group; a substitutedor unsubstituted aryl group; and a substituted or unsubstitutedheteroaryl group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In exemplary embodiments of Formula II, Ar is independently asubstituted pyridine oxide. For example, in exemplary embodiments ofFormula II, Ar is independently a substituted pyridine oxide selectedfrom a formula shown below,

where R₉ to R₁₂ is each independently selected from a group including,but not limited to, H; a halo (e.g., bromo, fluoro, chloro, iodo); analiphatic group; an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; anether; a substituted or unsubstituted amine; an ester; and an amidegroup.

In some embodiments, a subject ALDH agonist is a compound of genericformula Ia, as shown below:

where X_(n) and X_(y) are each independently H, C, N, O, or a halogen(e.g., F, Br, Cl, or I);

where . . . (dotted line) is an optional bond;

where z is the integer 0, 1, or 2, with the provisos that: 1) z=0 whenX=halogen and is not a bond; and 2) when z=0, X═O, . . . is not a bond,and one or more oxygen atoms (X) are present, oxygen is attached to amethyl group;

where n is the integer 0 or 1;

where y is the integer 0 or 1;

where A=C or S, and where a=1 when A=C; and where a=2 when A=S;

where Ar is independently a substituted pyridine oxide selected theformula from below,

where R₉ to R₁₂ is each independently selected from a group including,but not limit to, H; a halo (e.g., bromo, fluoro, chloro, iodo); analiphatic group; an alkyl group; a substituted alkyl group; an alkenylgroup; an alkynyl group; a substituted or unsubstituted cyclic group; anether; a substituted or unsubstituted amine; an ester; and an amidegroup;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

In some embodiments, a subject ALDH agonist has the structure of Formulaa, as shown below.

where R₁ to R₄ is each independently selected from, but not limited to,H; a halo (e.g., bromo, fluoro, chloro, iodo); an aliphatic group; analkyl group; a substituted alkyl group; an alkenyl group; an alkynylgroup; a substituted or unsubstituted cyclic group; an ether; asubstituted or unsubstituted amine; an ester or an amide group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

The following are exemplary, non-limiting compounds within Formula a.

In some embodiments, a subject ALDH agonist has the structure of Formulab, as shown below.

where R₁ to R₄ is each independently selected from, but not limit to, H;a halo (e.g., bromo, fluoro, chloro, iodo); an aliphatic group; an alkylgroup; a substituted alkyl group; an alkenyl group; an alkynyl group; asubstituted or unsubstituted cyclic group; an ether; a substituted orunsubstituted amine; an ester; and an amide group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

The following are exemplary, non-limiting compounds of Formula b:

In some embodiments, a subject ALDH agonist has the structure of Formulac, as shown below.

where R₁ to R₄ is each independently selected from such group, but notlimit to, H; a halo (e.g., bromo, fluoro, chloro, iodo); an aliphaticgroup; an alkyl group; a substituted alkyl group; an alkenyl group; analkynyl group; a substituted or unsubstituted cyclic group; an ether; asubstituted or unsubstituted amine; an ester; and an amide group;

or a pro-drug, a pharmaceutically acceptable salt, an analog, or aderivative thereof.

The following are exemplary, non-limiting compounds of Formula c:

Whether a compound is an ALDH agonist can be readily ascertained. Assaysfor dehydrogenase activity of ALDH are known in the art, and any knownassay can be used. Examples of dehydrogenase assays are found in variouspublications, including, e.g., Sheikh et al. ((1997) J. Biol. Chem.272:18817-18822); Vallari and Pietruszko (1984) J. Biol. Chem. 259:4922;and Farres et al. ((1994) J. Biol. Chem. 269:13854-13860).

As an example of an assay for dehydrogenase activity, ALDH aldehydedehydrogenase activity is assayed at 25° C. in 50 mM sodiumpyrophosphate HCl buffer, pH 9.0, 100 mM sodium phosphate buffer, pH7.4, or 50 mM sodium phosphate buffer, pH 7.4, where the buffer includesNAD⁺ (e.g., 0.8 mM NAD⁺, or higher, e.g., 1 mM, 2 mM, or 5 mM NAD⁺) andan aldehyde substrate such as 14 μM propionaldehyde. Reduction of NAD⁺is monitored at 340 nm using a spectrophotometer, or by fluorescenceincrease using a fluoromicrophotometer. Enzymatic activity can beassayed using a standard spectrophotometric method, e.g., by measuring areductive reaction of the oxidized form of nicotinamide adeninedinucleotide (NAD⁺) to its reduced form, NADH, at 340 nm, as describedin US 2005/0171043; and WO 2005/057213, and as depicted schematically inFIG. 4. In an exemplary assay, the reaction is carried out at 25° C. in0.1 sodium pyrophosphate (NaPPi) buffer, pH 9.0, 2.4 mM NAD⁺ and 10 mMacetaldehyde as the substrate. Enzymatic activity is measured by areductive reaction of NAD⁺ to NADH at 340 nm, as described in US2005/0171043; and WO 2005/057213. Alternatively, the production of NADHcan be coupled with another enzymatic reaction that consumes NADH andthat provides for a detectable signal. An example of such an enzymaticreaction is a diaphorase-based reaction, which reduces resazurin to itsoxidized fluorescent compound resorufin, as described in US2005/0171043; and WO 2005/057213, and as depicted schematically in FIG.4. Detection of fluorescent resorufin at 590 nm provides amplified andmore sensitive signals for any change in ALDH aldehyde dehydrogenaseenzymatic activity. NADP⁺ can be used in place of NAD⁺ in this assay. Insome embodiments, a substrate other than the substrate depicted in FIG.4 is used. Suitable substrates include, but are not limited to,octylaldehyde, phenylacetaldehyde, retinaldehyde, and 4-hydroxynonenal.Although the reaction depicted in FIG. 4 shows use of purified ALDH2,other ALDH polypeptides (e.g., ALDH1, ALDH3, ALDH5, etc.) can be used.The enzyme used in the assay can be purified (e.g., at least about 75%pure, at least about 80% pure, at least about 85% pure, at least about90% pure, at least about 95% pure, at least about 98% pure, or at leastabout 99% pure). Recombinant ALDH enzyme can also be used in the assay.

As another example, the effect of a compound on aldehyde dehydrogenaseactivity of an ALDH polypeptide can be assayed as described inWierzchowski et al. ((1996) Analytica Chimica Acta 319:209), in which afluorogenic synthetic substrate, e.g., 7-methoxy-1-naphthaldehyde isused. For example, the reaction could include7-methoxy-1-naphthaldehyde, NAD⁺, an ALDH polypeptide, and an ALDHagonist to be tested; fluorescence (excitation, 330 nm; emission 390 nm)is measured as a readout of enzymatic activity.

Whether a compound increases an esterase activity of ALDH can bedetermined using any known assay for esterase activity. For example,esterase activity of ALDH2 can be determined by monitoring the rate ofp-nitrophenol formation at 400 nm in 25 mMN,N-Bis(2-hydroxyethyl)-2-amino ethanesulfonic acid (BES) (pH 7.5) with800 μM p-nitrophenyl acetate as the substrate at room temperature in theabsence or presence of added NAD⁺. A pH-dependent molar extinctioncoefficient of 16 mM⁻¹ cm⁻¹ at 400 nm for nitrophenol can be used. See,e.g., Larson et al. (2007) J. Biol. Chem. 282:12940). Esterase activityof ALDH can be determined by measuring the rate of p-nitrophenolformation at 400 nm in 50 mM Pipes (pH 7.4) with 1 mMp-nitrophenylacetate as the substrate. A molar extinction coefficient of18.3×10³ M⁻¹ cm⁻¹ at 400 nm for p-nitrophenolate can be used forcalculating its rate of formation. See, e.g., Ho et al. (2005)Biochemistry 44:8022).

Whether a compound increases a reductase activity of ALDH can bedetermined using any known assay for reductase activity. A reductaseactivity of ALDH can be determined by measuring the rate of 1,2-glyceryldinitrate and 1,3-glyceryl dinitrate formation using a thin layerchromatography (TLC) or liquid scintillation spectrometry method, usinga radioactively labeled substrate. For example, 0.1 mM or 1 mM GTN(glyceryl trinitrate) is incubated with the assay mixture (1 ml)containing 100 mM KPi (pH 7.5), 0.5 mM EDTA, 1 mM NADH, 1 mM NADPH inthe presence ALDH2. After incubation at 37° C. for about 10 minutes toabout 30 minutes, the reaction is stopped and GTN and its metabolitesare extracted with 3×4 ml ether and pooled, and the solvent isevaporated by a stream of nitrogen. The final volume is kept to lessthan 100 ml in ethanol for subsequent TLC separation and scintillationcounting. See, e.g., Zhang and Stamler (2002) Proc. Natl. Acad. Sci. USA99:8306.

Pharmaceutical Compositions, Dosages, Routes of Administration

The present invention provides pharmaceutical compositions comprising asubject ALDH agonist. The terms “ALDH agonist” and “ALDH activator” arealso referred to herein as “active agent.” A subject ALDH agonist isformulated with one or more pharmaceutically acceptable excipients. Awide variety of pharmaceutically acceptable excipients are known in theart and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7^(th) ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3^(rd) ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectactive agent calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject active agent dependon the particular compound employed and the effect to be achieved, andthe pharmacodynamics associated with each compound in the host.

In the subject methods, a subject ALDH agonist may be administered tothe host using any convenient means capable of resulting in the desiredoutcome, e.g., reduction of disease, reduction of a symptom of adisease, etc. Thus, a subject ALDH agonist can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, a subject ALDH agonist can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

In pharmaceutical dosage forms, a subject ALDH agonist (“active agent”)may be administered in the form of its pharmaceutically acceptablesalts, or a subject active agent may be used alone or in appropriateassociation, as well as in combination, with other pharmaceuticallyactive compounds. The following methods and excipients are merelyexemplary and are in no way limiting.

For oral preparations, a subject active agent can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

A subject active agent can be formulated into preparations for injectionby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

A subject active agent can be utilized in aerosol formulation to beadministered via inhalation. A subject active agent can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject active agent can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. An active agent can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycol monomethyl ethers, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the subject active agent. Similarly, unit dosageforms for injection or intravenous administration may comprise a subjectactive agent in a composition as a solution in sterile water, normalsaline or another pharmaceutically acceptable carrier.

A subject active agent can be formulated for administration byinjection. Typically, injectable compositions are prepared as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection may also be prepared.The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles.

Ocular Formulations

A subject active agent can be formulated for ocular delivery, e.g.,where a subject active agent is formulated for delivery to the eye inliquid form (e.g., eye drops), or for injection into or around the eye.

A subject active agent can be formulated in an ophthalmic pharmaceuticalcomposition. Ophthalmic pharmaceutical compositions can be adapted fortopical administration to the eye in the form of solutions, suspensions,ointments, creams or as a solid insert. Ophthalmic formulations ofcomprising a subject active agent can contain from 0.01 to 5%, or from0.1 to 2% of a subject active agent. Higher dosages as, for example,about 10% or lower dosages can be employed provided the dose iseffective in treating a disorder of the eye (e.g., cataracts). For asingle dose, from between 0.001 to 5.0 mg, e.g., from 0.005 to 2.0 mg,or from 0.005 to 1.0 mg of a subject active agent can be applied to thehuman eye.

A subject active agent can be conveniently admixed with a non-toxicpharmaceutical organic carrier, or with a non-toxic pharmaceuticalinorganic carrier. Suitable pharmaceutically acceptable carriers are,for example, water, mixtures of water and water-miscible solvents suchas lower alkanols or aralkanols, vegetable oils, polyalkylene glycols,petroleum based jelly, ethyl cellulose, ethyl oleate,carboxymethyl-cellulose, polyvinylpyrrolidone, isopropyl myristate andother conventionally employed acceptable carriers. The pharmaceuticalpreparation can also contain non-toxic auxiliary substances such asemulsifying, preserving, wetting agents, bodying agents and the like, asfor example, polyethylene glycols 200, 300, 400 and 600, carbowaxes1,000, 1,500, 4,000, 6,000 and 10,000, antibacterial components such asquaternary ammonium compounds, phenylmercuric salts known to have coldsterilizing properties and which are non-injurious in use, thimerosal,methyl and propyl paraben, benzyl alcohol, phenyl ethanol, bufferingingredients such as sodium borate, sodium acetates, gluconate buffers,and other conventional ingredients such as sorbitan monolaurate,triethanolamine, oleate, polyoxyethylene sorbitan monopalmitate, dioctylsodium sulfosuccinate, monothioglycerol, thiosorbitol, ethylenediaminetetracetic acid, and the like.

Additionally, suitable ophthalmic vehicles can be used as carrier mediafor the present purpose including conventional phosphate buffer vehiclesystems, isotonic boric acid vehicles, isotonic sodium chloridevehicles, isotonic sodium borate vehicles and the like. Thepharmaceutical preparation can also be in the form of a microparticleformulation. The pharmaceutical preparation can also be in the form of asolid insert. For example, one may use a solid water soluble polymer asthe carrier for the medicament. The polymer used to form the insert canbe any water soluble non-toxic polymer, for example, cellulosederivatives such as methylcellulose, sodium carboxymethyl cellulose,(hydroxyloweralkyl cellulose), hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose; acrylates such as polyacrylicacid salts, ethylacrylates, polyacrylamides; natural products such asgelatin, alginates, pectins, tragacanth, karaya, chondrus, agar, acacia;the starch derivatives such as starch acetate, hydroxymethyl starchethers, hydroxypropyl starch, as well as other synthetic derivativessuch as polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methylether, polyethylene oxide, neutralized carbopol and xanthan gum, gellangum, and mixtures of said polymer.

A pharmaceutical preparation comprising a subject active agent canfurther include one or more non-toxic auxiliary substances such asantibacterial components which are non-injurious in use, for example,thimerosal, benzalkonium chloride, methyl and propyl paraben,benzyldodecinium bromide, benzyl alcohol, chlorhexidine, orphenylethanol; buffering ingredients such as sodium borate, sodiumacetate, sodium citrate, or gluconate buffers; and other conventionalingredients such as sodium chloride, sorbitan monolaurate,triethanolamine, polyoxyethylene sorbitan monopalmitate,ethylenediaminetetraacetic acid, and the like.

Topical Formulations

A subject active agent can be formulated for topical administration tothe skin. For example, a subject active agent can be formulated with oneor more dermatologically acceptable excipients.

The term “dermatologically acceptable,” as used herein, means that thecompositions or components thereof so described are suitable for use incontact with human skin without undue toxicity, incompatibility,instability, allergic response, and the like.

Suitable excipients include emollients; humectants; guanidine; glycolicacid and glycolate salts (e.g. ammonium and quaternary alkyl ammonium);lactic acid and lactate salts (e.g. ammonium and quaternary alkylammonium); aloe vera in any of its variety of forms (e.g., aloe veragel); sugar and starch derivatives (e.g., alkoxylated glucose);hyaluronic acid; lactamide monoethanolamine; acetamide monoethanolamine;and the like.

A variety of emollients may be employed to yield the conditioningcomponent of the present invention. These emollients may be selectedfrom one or more of the following classes: triglyceride esters thatinclude, but are not limited to, vegetable and animal fats and oils suchas castor oil, cocoa butter, safflower oil, cottonseed oil, corn oil,olive oil, cod liver oil, almond oil, avocado oil, palm oil, sesame oil,squalene, kikui oil and soybean oil; acetoglyceride esters, such asacetylated monoglycerides; alkyl esters of fatty acids having 10 to 20carbon atoms which include, but are not limited to, methyl, isopropyl,and butyl esters of fatty acids such as hexyl laurate, isohexyl laurate,isohexyl palmitate, isopropyl palmitate, decyloleate, isodecyl oleate,hexadecyl stearate decyl stearate, isopropyl isostearate, diisopropyladipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate,lauryl lactate, myristyl lactate, and cetyl lactate; alkenyl esters offatty acids having 10 to 20 carbon atoms such as oleyl myristate, oleylstearate, and oleyl oleate; fatty acids having 10 to 20 carbon atomssuch as pelargonic, lauric, myristic, palmitic, stearic, isostearic,hydroxystearic, oleic, linoleic, ricinoleic, arachidic, behenic, anderucic acids; fatty alcohols having 10 to 20 carbon atoms such aslauryl, myristyl, cetyl, hexadecyl, stearyl, isostearyl, hydroxystearyl,oleyl, ricinoleyl, behenyl, erucyl, and 2-octyl dodecanyl alcohols;fatty alcohol ethers such as propoxylated fatty alcohols of 10 to 20carbon atoms which include, but are not limited to, lauryl, cetyl,stearyl, isostearyl, oleyl, and cholesterol alcohols, having attachedthereto from 1 to 50 propylene oxide groups; lanolin and lanolinderivatives such as lanolin, lanolin oil, lanolin wax, lanolin alcohols,lanolin fatty acids, isopropyl lanolate, ethoxylated lanolin,ethoxylated lanolin alcohols, ethoxylated cholesterol, propoxylatedlanolin alcohols, acetylated lanolin alcohols, lanolin alcoholslinoleate, lanolin alcohols ricinoleate, acetate of lanolin alcoholsricinoleate, acetate of ethoxylated alcohols-esters, bydrogenolysis oflanolin, ethoxylated sorbitol lanolin, and liquid and semisolid lanolinabsorption bases; polyhydric alcohol esters such as ethylene glycolmono- and di-fatty acid esters, diethylene glycol mono- and di-fattyacid esters, polyethylene glycol (200-6000) mono- and di-fatty acidesters, propylene glycol mono- and di-fatty acid esters, polypropyleneglycol 2000 monooleate, polypropylene glycol 2000 monostearate, glycerylmono- and di-fatty acid esters, polyglycerol polyfatty esters,ethoxylated glyceryl monostearate, 1,2-butylene glycol monostearate,1,2-butylene glycol distearate, polyoxyethylene polyol fatty acid ester,sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acidesters; wax esters such as beeswax, spermaceti, myristyl myristate,stearyl stearate; forming a mixture of ether esters; and vegetable waxesincluding, but not limited to, camauba and candelilla waxes; andcholesterol fatty acid esters.

Humectants of the polyhydric alcohol-type are suitable for use. Typicalpolyhydric alcohols include polyalkylene glycols and more preferablyalkylene polyols and their derivatives, including propylene glycol,dipropylene glycol, tripropylene glycol, polyethylene glycol andderivatives thereof, sorbitol, hydroxypropyl sorbitol, erythritol,threitol, pentaerythritol, xylitol, glucitol, mannitol, hexylene glycol,1,3-butylene glycol, 1,2,6-hexanetriol, glycerol, ethoxylated glycerol,propoxylated glycerol, sodium 2-pyrrolidone-5-carboxylate, solublecollagen, gelatin and mixtures thereof.

Also useful herein are guanidine; glycolic acid and glycolate salts(e.g. ammonium and quaternary alkyl ammonium); lactic acid and lactatesalts (e.g. ammonium and quaternary alkyl ammonium); aloe vera in any ofits variety of forms (e.g., aloe vera gel); sugar and starch derivatives(e.g., alkoxylated glucose); hyaluronic acid; lactamidemonoethanolamine; acetamide monoethanolamine; and mixtures thereof.

A composition comprising a subject active agent can include adermatologically-acceptable hydrophilic diluent. Non-limiting examplesof hydrophilic diluents are water, organic hydrophilic diluents such aslower monovalent alcohols (e.g., C₁-C₄ alcohols) and low molecularweight glycols and polyols, including propylene glycol, polyethyleneglycol (e.g., Molecular Weight 200-600 g/mole), polypropylene glycol(e.g., Molecular Weight 425-2025 g/mole), glycerol, butylene glycol,1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol,isopropanol, sorbitol esters, butanediol, ether propanol, ethoxylatedethers, propoxylated ethers and combinations thereof. A compositioncomprising a subject active agent can contain from about 60% to about99.99% of a hydrophilic diluent.

A composition comprising a subject active agent can include adermatologically acceptable carrier. An example of a suitable carrier isan emulsion comprising a hydrophilic phase comprising a hydrophiliccomponent, e.g., water or other hydrophilic diluent, and a hydrophobicphase comprising a hydrophobic component, e.g., a lipid, oil or oilymaterial. The hydrophilic phase will be dispersed in the hydrophobicphase, or vice versa, to form respectively hydrophilic or hydrophobicdispersed and continuous phases, depending on the compositioningredients. In emulsion technology, the term “dispersed phase” is aterm well known to one skilled in the art which means that the phaseexists as small particles or droplets that are suspended in andsurrounded by a continuous phase. The dispersed phase is also known asthe internal or discontinuous phase. The emulsion may be or comprise(e.g., in a triple or other multi-phase emulsion) an oil-in-wateremulsion or a water-in-oil emulsion such as a water-in-siliconeemulsion. Oil-in-water emulsions can comprise from about 1% to about 50%of the dispersed hydrophobic phase and from about 1% to about 98% of thecontinuous hydrophilic phase; water-in-oil emulsions can comprise fromabout 1% to about 98% of the dispersed hydrophilic phase and from about1% to about 50% of the continuous hydrophobic phase.

A subject active agent can be formulated with common excipients,diluents, or carriers, and formed into lotions, creams, solutions,suspensions, powders, aerosols, emulsions, salves, ointments and thelike. Examples of excipients, diluents, and carriers that are suitablefor such formulations include buffers, as well as fillers and extenderssuch as starch, cellulose, sugars, mannitol, and silicic derivatives.Binding agents can also be included such as carboxymethyl cellulose,hydroxymethylcellulose, hydroxypropyl methylcellulose and othercellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone.Moisturizing agents can be included such as glycerol, disintegratingagents such as calcium carbonate and sodium bicarbonate. Agents forretarding dissolution can also be included such as paraffin. Resorptionaccelerators such as quaternary ammonium compounds can also be included.Surface active agents such as cetyl alcohol and glycerol monostearatecan be included. Adsorptive carriers such as kaolin and bentonite can beadded. Lubricants such as talc, calcium and magnesium stearate, andsolid polyethyl glycols can also be included. Preservatives may also beadded. A composition comprising a subject ALDH agonist can includethickening agents such as cellulose and/or cellulose derivatives. Acomposition comprising a subject ALDH agonist can include contain gumssuch as xanthan, guar or carbo gum or gum arabic, or alternativelypoly(ethylene glycol)s, bentones and montmorillonites, and the like.

A composition comprising a subject ALDH agonist can further include oneor more additional agents such as, for example, antioxidants,surfactants, preservatives, film-forming, keratolytic or comedolyticagents, perfumes, flavorings, and colorings. Antioxidants such ast-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytolueneand α-tocopherol and its derivatives can be added.

Furthermore, composition comprising a subject ALDH agonist can furtherinclude one or more additional therapeutic agents, for example,anti-microbial agents, pain relievers, anti-inflammatory agents, and thelike, depending, e.g., on the condition being treated.

Continuous Delivery

In some embodiments, a subject active agent is delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of active agent can be accomplished usingany of a variety of refillable, pump systems. Pumps provide consistent,controlled release over time. In some embodiments, the agent is in aliquid formulation in a drug-impermeable reservoir, and is delivered ina continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the invention may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present invention.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT publishedapplication no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and5,728,396)). Exemplary osmotically-driven devices suitable for use inthe invention include, but are not necessarily limited to, thosedescribed in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426;3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202;4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850;4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692;5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted infra, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, an active agent is delivered using an implantabledrug delivery system, e.g., a system that is programmable to provide foradministration of a subject active agent. Exemplary programmable,implantable systems include implantable infusion pumps. Exemplaryimplantable infusion pumps, or devices useful in connection with suchpumps, are described in, for example, U.S. Pat. Nos. 4,350,155;5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704;6,464,687; 6,475,180; and 6,512,954. A further exemplary device that canbe adapted for the present invention is the Synchromed infusion pump(Medtronic).

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

Oral Formulations

In some embodiments, a subject active agent is formulated for oraldelivery to an individual in need of such an agent.

For oral delivery, a subject formulation comprising a subject activeagent will in some embodiments include an enteric-soluble coatingmaterial. Suitable enteric-soluble coating material includehydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropylmethyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),polyvinyl phthalic acetate (PVPA), Eudragit™, and shellac.

As one non-limiting example of a suitable oral formulation, a subjectactive agent is formulated with one or more pharmaceutical excipientsand coated with an enteric coating, as described in U.S. Pat. No.6,346,269. For example, a solution comprising a subject active agent anda stabilizer is coated onto a core comprising pharmaceuticallyacceptable excipients, to form an active agent-coated core; asub-coating layer is applied to the active agent-coated core, which isthen coated with an enteric coating layer. The core generally includespharmaceutically inactive components such as lactose, a starch,mannitol, sodium carboxymethyl cellulose, sodium starch glycolate,sodium chloride, potassium chloride, pigments, salts of alginic acid,talc, titanium dioxide, stearic acid, stearate, micro-crystallinecellulose, glycerin, polyethylene glycol, triethyl citrate, tributylcitrate, propanyl triacetate, dibasic calcium phosphate, tribasic sodiumphosphate, calcium sulfate, cyclodextrin, and castor oil. Suitablesolvents for a subject active agent include aqueous solvents. Suitablestabilizers include alkali-metals and alkaline earth metals, bases ofphosphates and organic acid salts and organic amines. The sub-coatinglayer comprises one or more of an adhesive, a plasticizer, and ananti-tackiness agent. Suitable anti-tackiness agents include talc,stearic acid, stearate, sodium stearyl fumarate, glyceryl behenate,kaolin and aerosil. Suitable adhesives include polyvinyl pyrrolidone(PVP), gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose(HPC), hydroxypropyl methyl cellulose (HPMC), vinyl acetate (VA),polyvinyl alcohol (PVA), methyl cellulose (MC), ethyl cellulose (EC),hydroxypropyl methyl cellulose phthalate (HPMCP), cellulose acetatephthalates (CAP), xanthan gum, alginic acid, salts of alginic acid,Eudragit™, copolymer of methyl acrylic acid/methyl methacrylate withpolyvinyl acetate phthalate (PVAP). Suitable plasticizers includeglycerin, polyethylene glycol, triethyl citrate, tributyl citrate,propanyl triacetate and castor oil. Suitable enteric-soluble coatingmaterial include hydroxypropyl methylcellulose acetate succinate(HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalate (CAP), polyvinyl phthalic acetate (PVPA), Eudragit™and shellac.

Suitable oral formulations also include a subject active agentformulated with any of the following: microgranules (see, e.g., U.S.Pat. No. 6,458,398); biodegradable macromers (see, e.g., U.S. Pat. No.6,703,037); biodegradable hydrogels (see, e.g., Graham and McNeill(1989) Biomaterials 5:27-36); biodegradable particulate vectors (see,e.g., U.S. Pat. No. 5,736,371); bioabsorbable lactone polymers (see,e.g., U.S. Pat. No. 5,631,015); slow release protein polymers (see,e.g., U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); apoly(lactide-co-glycolide/polyethylene glycol block copolymer (see,e.g., U.S. Pat. No. 6,630,155; Atrix Laboratories, Inc.); a compositioncomprising a biocompatible polymer and particles of metalcation-stabilized agent dispersed within the polymer (see, e.g., U.S.Pat. No. 6,379,701; Alkermes Controlled Therapeutics, Inc.); andmicrospheres (see, e.g., U.S. Pat. No. 6,303,148; Octoplus, B.V.).

Suitable oral formulations also include a subject active agent with anyof the following: a carrier such as Emisphere® (Emisphere Technologies,Inc.); TIMERx, a hydrophilic matrix combining xanthan and locust beangums which, in the presence of dextrose, form a strong binder gel inwater (Penwest); Geminex™ (Penwest); Procise™ (GlaxoSmithKline); SAVIT™(Mistral Pharma Inc.); RingCap™ (Alza Corp.); Smartrix® (SmartrixTechnologies, Inc.); SQZgel™ (MacroMed, Inc.); Geomatrix™ (Skye Pharma,Inc.); Oros® Tri-layer (Alza Corporation); and the like.

Also suitable for use are formulations such as those described in U.S.Pat. No. 6,296,842 (Alkermes Controlled Therapeutics, Inc.); U.S. Pat.No. 6,187,330 (Scios, Inc.); and the like.

Also suitable for use herein are formulations comprising an intestinalabsorption enhancing agent. Suitable intestinal absorption enhancersinclude, but are not limited to, calcium chelators (e.g., citrate,ethylenediamine tetracetic acid); surfactants (e.g., sodium dodecylsulfate, bile salts, palmitoylcarnitine, and sodium salts of fattyacids); toxins (e.g., zonula occludens toxin); and the like.

Inhalational Formulations

A subject ALDH agonist will in some embodiments be administered to apatient by means of a pharmaceutical delivery system for the inhalationroute. A subject ALDH agonist can be formulated in a form suitable foradministration by inhalation. The inhalational route of administrationprovides the advantage that the inhaled drug can bypass the blood-brainbarrier. The pharmaceutical delivery system is one that is suitable forrespiratory therapy by delivery of a subject ALDH agonist to mucosallinings of the bronchi. This invention can utilize a system that dependson the power of a compressed gas to expel the subject ALDH agonist froma container. An aerosol or pressurized package can be employed for thispurpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed in this invention, the aerosolcontains a subject ALDH agonist, which can be dissolved, suspended, oremulsified in a mixture of a fluid carrier and a propellant. The aerosolcan be in the form of a solution, suspension, emulsion, powder, orsemi-solid preparation. Aerosols employed in the present invention areintended for administration as fine, solid particles or as liquid mistsvia the respiratory tract of a patient. Various types of propellantsknown to one of skill in the art can be utilized. Suitable propellantsinclude, but are not limited to, hydrocarbons or other suitable gas. Inthe case of the pressurized aerosol, the dosage unit may be determinedby providing a value to deliver a metered amount.

A subject ALDH agonist can also be formulated for delivery with anebulizer, which is an instrument that generates very fine liquidparticles of substantially uniform size in a gas. For example, a liquidcontaining the subject ALDH agonist is dispersed as droplets. The smalldroplets can be carried by a current of air through an outlet tube ofthe nebulizer. The resulting mist penetrates into the respiratory tractof the patient.

A powder composition containing a subject ALDH agonist, with or withouta lubricant, carrier, or propellant, can be administered to a mammal inneed of therapy. This embodiment of the invention can be carried outwith a conventional device for administering a powder pharmaceuticalcomposition by inhalation. For example, a powder mixture of the compoundand a suitable powder base such as lactose or starch may be presented inunit dosage form in for example capsular or cartridges, e.g. gelatin, orblister packs, from which the powder may be administered with the aid ofan inhaler.

There are several different types of inhalation methodologies which canbe employed in connection with the present invention. A subject ALDHagonist can be formulated in basically three different types offormulations for inhalation. First, a subject ALDH agonist can beformulated with low boiling point propellants. Such formulations aregenerally administered by conventional meter dose inhalers (MDI's).However, conventional MDI's can be modified so as to increase theability to obtain repeatable dosing by utilizing technology whichmeasures the inspiratory volume and flow rate of the patient asdiscussed within U.S. Pat. Nos. 5,404,871 and 5,542,410.

Alternatively, a subject ALDH agonist can be formulated in aqueous orethanolic solutions and delivered by conventional nebulizers. In someembodiments, such solution formulations are aerosolized using devicesand systems such as disclosed within U.S. Pat. Nos. 5,497,763;5,544,646; 5,718,222; and 5,660,166.

A subject ALDH agonist can be formulated into dry powder formulations.Such formulations can be administered by simply inhaling the dry powderformulation after creating an aerosol mist of the powder. Technology forcarrying such out is described within U.S. Pat. No. 5,775,320 and U.S.Pat. No. 5,740,794.

Dosages and Dosing

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus, for example, oral dosages may be about ten times the injectiondose. Higher doses may be used for localized routes of delivery.

For example, a subject ALDH activity modulator can be administered in anamount of from about 1 mg to about 1000 mg per dose, e.g., from about 1mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg toabout 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100mg, from about 100 mg to about 125 mg, from about 125 mg to about 150mg, from about 150 mg to about 175 mg, from about 175 mg to about 200mg, from about 200 mg to about 225 mg, from about 225 mg to about 250mg, from about 250 mg to about 300 mg, from about 300 mg to about 350mg, from about 350 mg to about 400 mg, from about 400 mg to about 450mg, from about 450 mg to about 500 mg, from about 500 mg to about 750mg, or from about 750 mg to about 1000 mg per dose.

An exemplary dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is in some embodiments one whichprovides up to about 1 μg to about 1,000 μg or about 10,000 μg ofsubject compound in a blood sample taken from the individual beingtreated, about 24 hours after administration of the compound to theindividual.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound(s) in a compositionas a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

In some embodiments, multiple doses of a subject compound areadministered. The frequency of administration of a subject compound(“active agent”) can vary depending on any of a variety of factors,e.g., severity of the symptoms, etc. For example, in some embodiments, asubject compound is administered once per month, twice per month, threetimes per month, every other week (qow), once per week (qw), twice perweek (biw), three times per week (tiw), four times per week, five timesper week, six times per week, every other day (qod), daily (qd), twice aday (bid), or three times a day (tid). As discussed above, in someembodiments, a subject compound is administered continuously.

The duration of administration of a subject compound, e.g., the periodof time over which a subject compound is administered, can vary,depending on any of a variety of factors, e.g., patient response, etc.For example, a subject compound can be administered over a period oftime ranging from about one day to about one week, from about two weeksto about four weeks, from about one month to about two months, fromabout two months to about four months, from about four months to aboutsix months, from about six months to about eight months, from abouteight months to about 1 year, from about 1 year to about 2 years, orfrom about 2 years to about 4 years, or more. In some embodiments, asubject compound is administered for the lifetime of the individual.

Routes of Administration

A subject ALDH agonist is administered to an individual using anyavailable method and route suitable for drug delivery, including in vivoand ex vivo methods, as well as systemic and localized routes ofadministration. Administration can be acute (e.g., of short duration,e.g., a single administration, administration for one day to one week),or chronic (e.g., of long duration, e.g., administration for longer thanone week, e.g., administration over a period of time of from about 2weeks to about one month, from about one month to about 3 months, fromabout 3 months to about 6 months, from about 6 months to about 1 year,or longer than one year).

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, transdermal, sublingual, topical application, intravenous,ocular, rectal, nasal, oral, and other enteral and parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the agent and/or the desired effect. Thecompound can be administered in a single dose or in multiple doses.

A subject active agent can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, and inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, ocular, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofthe agent. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of a subject ALDH agonist through the skin ormucosa include, but are not necessarily limited to, topical applicationof a suitable pharmaceutical preparation, transdermal transmission,injection and epidermal administration. For transdermal transmission,absorption promoters or iontophoresis are suitable methods.Iontophoretic transmission may be accomplished using commerciallyavailable “patches” which deliver their product continuously viaelectric pulses through unbroken skin for periods of several days ormore.

Treatment Methods

The present invention provides various treatment methods, generallyinvolving administering to an individual in need thereof an effectiveamount of a subject agonist. A subject ALDH agonist is suitable fortreating a variety of disorders, including, e.g., conditions involvingischemic stress, chronic free-radical associated diseases, acutefree-radical associated diseases, insensitivity to nitroglycerin (e.g.,in angina and heart failure), hypertension, cataracts, diabetes, andosteoporosis. A subject ALDH agonist is suitable for sensitizing acancerous cell to a cancer chemotherapeutic agent or other standardcancer therapy; for treating alcohol (e.g., ethanol; ethyl alcohol)addiction; and for treating narcotic addiction.

Methods of Treating Conditions Involving Ischemic Stress

The present invention provides methods for treating conditions involvingischemic stress, including prophylactic methods, in an individual, themethods generally involving administering to an individual in needthereof an effective amount of a subject ALDH agonist. Conditionsinvolving ischemic stress include ischemic conditions, ischemic events,conditions that can give rise to ischemia, and conditions that resultfrom an ischemic event. Conditions involving ischemic stress that areamenable to treatment with a subject method include ischemia that resultfrom any condition or event, including, but not limited to, myocardialinfarct (e.g., acute myocardial infarction), cardiac surgery, braintrauma, cerebrovascular disease, stroke, spinal cord injury,subarachnoid hemorrhage, major surgery in which ischemia to variety oforgans occur, organ transplantation, limb ischemia (e.g., resulting fromType 1 or Type 2 diabetes), and the like.

In some embodiments, the agent is administered before a predicted oranticipated ischemic event, e.g., from about 1 hour to about 1 weekbefore the ischemic event, e.g., from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 8hours, from about 8 hours to about 12 hours, from about 12 hours toabout 16 hours, from about 16 hours to about 24 hours, from about 24hours to about 36 hours, from about 36 hours to about 48 hours, fromabout 48 hours to about 72 hours, or from about 72 hours to about 1 weekpreceding the predicted or anticipated ischemic event.

Pretreatment with an active agent is desirable under certaincircumstances, for example, when a subject has already experienced astroke, when a subject is about to undergo cardiac surgery, etc. Forexample, a patient who has already experienced a stroke will have anincreased probability of experiencing a second stroke. Subjects who aresusceptible to transient ischemic attacks also have an increased risk ofa stroke. Subjects who suffer a subarachnoid hemorrhage may experiencefurther ischemic events induced by vasospasms that constrict the bloodvessels. Subjects who experience trauma to organs such as the brain arealso susceptible to an ischemic event. Subjects undergoing surgery overan extended period of time are also susceptible to an ischemic event.The above situations exemplify circumstances when a subject wouldbenefit from pretreatment with a subject ALDH agonist.

In some embodiments, a subject ALDH agonist is administered after anischemic event. For example, a subject ALDH agonist is effective inreducing the adverse effects of an ischemic event such as cardiacischemia, reperfusion injury, cerebrovascular disease, acute myocardialinfarction, subarachnoid hemorrhage, and trauma. In some embodiments, asubject ALDH agonist is administered within 1 minute to within 15 hours,e.g., from about 1 minute to about 5 minutes, from about 5 minutes toabout 10 minutes, from about 10 minutes to about 15 minutes, from about15 minutes to about 30 minutes, from about 30 minutes to about 60minutes, from about 60 minutes to about 2 hours, from about 2 hours toabout 4 hours, from about 4 hours to about 8 hours, from about 8 hoursto about 12 hours, or from about 12 hours to about 15 hours, followingthe ischemic event. In some embodiments, an increased concentration of asubject ALDH2 agonist is maintained in the plasma for at least severalhours to several days following the ischemic event.

For example, in some embodiments, a subject ALDH agonist is administeredto an individual who has suffered an acute myocardial infarction (AMI)within 1 minute to within 15 hours, e.g., from about 1 minute to about 5minutes, from about 5 minutes to about 10 minutes, from about 10 minutesto about 15 minutes, from about 15 minutes to about 30 minutes, fromabout 30 minutes to about 60 minutes, from about 60 minutes to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about8 hours, from about 8 hours to about 12 hours, or from about 12 hours toabout 15 hours, following the AML

Methods of Treating Ocular Disorders

The present invention provides methods for treating ocular disorders,e.g., cataracts, the methods generally involving administering to anindividual in need thereof an effective amount of a subject ALDHagonist. In some embodiments, the ALDH agonist will be an ALDH3 agonist.The ALDH agonist will be formulated for ocular administration, e.g., fortopical administration to the eye, for injection into the eye (e.g.,intravitreal injection), or some other route of administration to theeye. Ocular disorders that can be treated with a subject ALDH agonistinclude, e.g., age-related cataracts, secondary cataracts, traumaticcataracts, congenital cataracts, radiation cataracts, etc.

Among the risk factors for cataract are exposure to UV-light (which canresult in generation of toxic aldehydes such as 4-hydroxy-2-nonenal),exposure to cigarette smoke (cigarette smoke contains high amounts ofreactive aldehdyes, such as acrolein). See, e.g., Jia et al., InvestOphthalmol Vis Sci. 2007 January; 48(1):339-48. PMID: 17197552; J Donget al., Neurochem. 2007 November; 103(3):1041-52. PMID: 17935603; Papaet al., Free Radic Biol Med. 2003 May 1; 34(9):1178-89. PMID: 12706498;King et al. J Exp Zool. 1998 Sep.-Oct. 1; 282(1-2):12-7. PMID: 9723161).The instant disclosure provides methods of treating cataracts, themethods generally involving administering to an individual in needthereof an effective amount of an ALDH agonist, e.g., a subject ALDHagonist.

A subject ALDH agonist can be administered to an individual in needthereof for the treatment of an ocular disorder (e.g., cataracts), wherethe ALDH agonist is administered topically to the eye, e.g., in the formof eyedrops.

A subject ALDH agonist can be administered once per month, twice permonth, three times per month, every other week (qow), once per week(qw), twice per week (biw), three times per week (tiw), four times perweek, five times per week, six times per week, every other day (qod),daily (qd), twice a day (bid), or three times a day (tid), for thetreatment of an ocular disorder. A subject ALDH agonist can beadministered over a period of time of from about 3 months to about 1year, from 1 year to 10 years, or more than 10 years.

In some embodiments, where a subject ALDH agonist is administered forthe treatment of cataracts, the ALDH agonist is administered before orafter surgery for cataracts.

Methods of Treating Skin Disorders

The present invention provides methods for treating skin disorders, themethods generally involving administering to an individual in needthereof an effective amount of a subject ALDH agonist.

Skin disorders that can be treated with a subject ALDH agonist include,but are not limited to, dermatitis. In some embodiments, for thetreatment of a skin disorder, a subject ALDH agonist is administeredtopically to the skin, e.g., to an area of skin affected by a skindisorder.

Methods of Treating Chronic and Acute Free-Radical Associated Diseases

The present invention provides methods for treating acute and chronicfree-radical associated diseases in an individual, the methods generallyinvolving administering to an individual in need thereof an effectiveamount of a subject ALDH agonist.

Acute Free-Radical Associated Disorders

The present invention provides methods for treating acute free-radicalassociated diseases in an individual, the methods generally involvingadministering to an individual in need thereof an effective amount of asubject ALDH agonist. Acute free radical-associated disorders that areamenable to treatment with a subject method include seizures (Patel etal. (2001) Journal of Neurochemistry 79:1065-1069); skin damageresulting from UV exposure, and photodamage of skin (e.g., “sunburn”)(Aldini et al. (2007) Chem Res Toxicol. 20(3):416-23); acute thermalskin burn injury (Pintaudi et al. (2000) Free Radical Res.33(2):139-46); and tissue hyperoxia (e.g., hyperoxia-induced chroniclung disease; and bronchopulmonary dysplasia) (Xu et al. (2006) Am J.Phsiol. Lung Cell. Mol. Physiol. 291(5):L966-75).

The present invention provides methods for treating sunburn in anindividual, the methods generally involving administering to anindividual in need thereof an effective amount of a subject ALDHagonist. In some embodiments, a subject method for treating sunburncomprises topically applying a formulation comprising a subject ALDHagonist to an area of the skin affected by sunburn.

The present invention provides methods for treating a seizure in anindividual, the methods generally involving administering to anindividual in need thereof an effective amount of a subject ALDHagonist. In some embodiments, a subject ALDH agonist is administeredafter a seizure has occurred, e.g., within from about 1 minute to about5 minutes, from about 5 minutes to about 15 minutes, from about 15minutes to about 30 minutes, from about 30 minutes to about 1 hour, orfrom about 1 hour to about 4 hours following a seizure. In otherembodiments, a subject ALDH agonist is administered prophylactically,e.g., a subject ALDH agonist is administered to an individual who hasexperienced a seizure in the past, to reduce the likelihood that anotherseizure will occur. In some embodiments, an effective amount of asubject ALDH agonist is an amount that is effective to reduce at leastone of the severity of a seizure, the frequency of seizures, and theduration of a seizure.

Chronic Free-Radical Associated Diseases

The present invention provides methods for treating chronic free-radicalassociated diseases in an individual, the methods generally involvingadministering to an individual in need thereof an effective amount of asubject ALDH agonist. Chronic free radical-associated disorders that areamenable to treatment with a subject method include neurodegenerativediseases such as Parkinson's Disease and Alzheimer's Disease (Burke etal. (2003) Neurol. Dis. 2(2):143; and Ohta and Ohsawa (2006) J.Alzheimer's Disease 9(2):155); amyotrophic lateral sclerosis (ALS);cancer such as esophageal cancer (Chen et al. (2006) Int J Cancer2119(12):2827-31); upper aerodigestive tract cancer (Hashibe et al.(2006) Cancer Epidemiol Biomarkers Prev. 15(4):696-703); head and necksquamous cell carcinoma (Hashimoto et al. (2006) Tumour Biol.27(6):334-8; Yokoyama et al. (2005) Alcohol. 35(3):175-85);cardiovascular diseases such as atherosclerosis (Narita et al. (2003)Ultrasound in Medicine and Biology 29(10):1415-1419); and the like. Insome embodiments, a chronic free radical-associated disease is treatedby chronic (e.g., daily) treatment with a subject ALDH agonist.

The present invention provides a method for treating Alzheimer's Disease(AD) in an individual suffering from AD, the method generally involvingadministering to the individual an effective amount of a subject ALDHagonist. In some embodiments, an “effective amount” of a subject ALDHagonist is an amount that is effective to at least slow the decline incognitive function in the individual. In some embodiments, an “effectiveamount” of a subject ALDH agonist is an amount that is effective toimprove memory in the individual being treated. In some embodiments, asubject ALDH agonist is administered to the individual systemically,over a period of time of from about 3 months to about 6 months, fromabout 6 months to about 1 year, or more than 1 year.

The present invention provides a method for treating Parkinson's Diseasein an individual, the method generally involving administering to theindividual an effective amount of a subject ALDH agonist. In someembodiments, an “effective amount” of a subject ALDH agonist is anamount that is effective to ameliorate one or more symptoms ofParkinson's Disease. In some embodiments, an “effective amount” of asubject ALDH agonist is an amount that is effective to slow the progressof the disease. In some embodiments, a subject ALDH agonist isadministered to the individual systemically, over a period of time offrom about 3 months to about 6 months, from about 6 months to about 1year, or more than 1 year.

Methods of Treating Heart Conditions

The present invention provides methods of treating disorders such asangina, heart failure, insensitivity to nitroglycerin in angina andheart failure (Li et al. (2006) J. Clin. Invest. 116:506-511),hypertension (Asselin et al. (2006) Free Radical Biol. and Med. 41:97),and heart disease. The methods generally involve administering to anindividual in need thereof an effective amount of a subject ALDHagonist.

In some embodiments, a subject ALDH agonist is administered to anindividual in conjunction with nitroglycerin treatment. The subject ALDHagonist and the nitroglycerin can be administered by the same route ofadministration (e.g., oral, sublingual, transdermal, translingual,etc.). In the alternative, subject ALDH agonist and the nitroglycerincan be administered by different routes of administration. For example,in some embodiments, nitroglycerin is administered sublingually,translingually, transdermally, or orally; and a subject ALDH agonist isadministered via a different route of administration (e.g., intravenous,intramuscular, etc.). The ALDH agonist can be administered before,during, or after administration of the nitroglycerin.

An effective amount of a subject ALDH agonist is an amount that, whenadministered in combination therapy with nitroglycerin, is effective toreduce angina by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%, or more,within a period of time of from about 1 minute to about 2 minutes, fromabout 2 minutes to about 3 minutes, from about 3 minutes to about 4minutes, from about 4 minutes to about 5 minutes, or from about 5minutes to about 10 minutes, following administration of the ALDHagonist. In some embodiments, a subject ALDH agonist and nitroglycerinare administered substantially simultaneously, e.g., within about twominutes, within about 1 minute, or within about 30 seconds of oneanother. The term “combination therapy with nitroglycerin” encompassesadministration of a subject ALDH agonist substantially simultaneouslywith nitroglycerin; administration of a subject ALDH agonist beforeadministration of nitroglycerin; administration of a subject ALDHagonist after administration of nitroglycerin; etc.

In some embodiments, an effective amount of a subject ALDH agonist is anamount that is effective to treat hypertension, e.g., to reduce one ormore symptoms or indications of hypertension in an individual. Forexample, in some embodiments, an effective amount of a subject ALDHagonist is an amount that is effective to reduce blood pressure in theindividual by at least about 5%, at least about 10%, at least about 15%,at least about 20%, or at least about 25%, or more, or to bring theblood pressure of the individual to within a normal range.

In some embodiments, an effective amount of a subject ALDH agonist is anamount that is effective to treat heart disease, e.g., to reduce one ormore symptoms or indications of heart disease in an individual. Whethera given ALDH agonist is effective to treat heart disease can bedetermined using standard methods of assessing heart function, e.g.,electrocardiogram, angiogram, and the like.

Methods of Detoxification

The present invention provides methods of reducing the levels of a toxiccompound in an individual, the methods generally involving administeringto an individual in need thereof an effective amount of a subject ALDHagonist. The present invention provides methods of treating a disorderassociated with or resulting from a toxic level of a compound (e.g., axenogenic aldehyde; a biogenic aldehyde; or a compound that, wheningested, absorbed, or inhaled, gives rise to an aldehyde substrate forALDH), the methods generally involving administering to an individual inneed thereof an effective amount of a subject ALDH agonist, where thelevel of the compound in the individual is reduced to a non-toxic level.

Toxic compounds whose levels can be reduced in an individual using asubject method include, but are not limited to, ethanol, methanol,ethylene glycol monomethyl ether, xenogenic aldehydes, biogenicaldehydes, and an aldehyde produced by in vivo metabolism of a compoundthat is ingested, absorbed, or inhaled. A subject ALDH agonist isadministered in an amount that is effective, when administered in one ormore doses, to reduce a toxic level of a compound such as ethanol,methanol, ethylene glycol monomethyl ether, xenogenic aldehydes,biogenic aldehydes, or an aldehyde produced by in vivo metabolism of acompound that is ingested, absorbed, or inhaled. In some embodiments,the aldehyde is acetaldehyde.

As an example, a subject ALDH agonist is administered to an individualfollowing excessive alcohol (e.g., ethanol) consumption; and toxiclevels of alcohol or aldehyde (e.g., an aldehyde that is a metabolicproduct of ethanol) in the individual are reduced by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or at least about 90%, or more, compared to the alcohol or aldehydelevels in the individual before treatment with the ALDH agonist. In someembodiments, a subject ALDH agonist is administered in an amount that iseffective to reduce a toxic alcohol or aldehyde level by at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90%, or more, within from about 5 minutes toabout 15 minutes, from about 15 minutes to about 30 minutes, from about30 minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH agonist. In some embodiments, a subject ALDH agonist isadministered in an amount that is effective to reduce a toxic alcohol oraldehyde level to a non-toxic level within from about 5 minutes to about15 minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH agonist.

As an example, a subject ALDH agonist is administered to an individualfollowing excessive alcohol (e.g., ethanol) consumption; and levels ofacetaldehyde in the individual are reduced by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, or more, compared to the alcohol or aldehyde levels inthe individual before treatment with the ALDH agonist. In someembodiments, a subject ALDH agonist is administered in an amount that iseffective to reduce an acetaldehyde level by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, or more, within from about 5 minutes to about 15minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH agonist.

The present invention provides methods of reducing aldehyde toxicity,the methods generally involving administering an effective amount of asubject ALDH agonist. In some embodiments, an effective amount of anALDH agonist is an amount that is effective to reduce one or moresymptoms of aldehyde toxicity. For example, in some embodiments, aneffective amount of an ALDH agonist is an amount that is effective toreduce one or more symptoms of excess ethanol consumption, where suchsymptoms include, e.g., headache, dehydration, fatigue, nausea,vomiting, diarrhea, weakness, anxiety, irritability, photophobia,phonophobia, etc.

As an example, a subject ALDH agonist is administered to an individualhaving a toxic level of an aldehyde (e.g., following excessive ethanolconsumption); and toxic levels of an aldehyde in the individual arereduced by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or at least about 90%, or more, comparedto the level of the aldehyde in the individual before treatment with theALDH agonist. In some embodiments, a subject ALDH agonist isadministered in an amount that is effective to reduce a toxic aldehydelevel by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%, or more, within fromabout 5 minutes to about 15 minutes, from about 15 minutes to about 30minutes, from about 30 minutes to 1 hour, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH agonist. In some embodiments, a subject ALDHagonist is administered in an amount that is effective to reduce a toxicaldehyde level to a non-toxic level within from about 5 minutes to about15 minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH agonist.

In some embodiments, a subject ALDH agonist reduces the level of bothethanol and an aldehyde, e.g., following excessive ethanol consumption,as described above.

As another example, a subject ALDH agonist is administered to anindividual having toxic levels of methanol or ethylene glycol monomethylether; and the toxic level of methanol or ethylene glycol monomethylether is reduced by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, or more,compared to the methanol or ethylene glycol monomethyl ether level inthe individual before treatment with the ALDH agonist. In someembodiments, a subject ALDH agonist is administered in an amount that iseffective to reduce a toxic methanol or ethylene glycol monomethyl etherlevel by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%, or more, within fromabout 5 minutes to about 15 minutes, from about 15 minutes to about 30minutes, from about 30 minutes to 1 hour, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH agonist. In some embodiments, a subject ALDHagonist is administered in an amount that is effective to reduce a toxicmethanol or ethylene glycol monomethyl ether level to a non-toxic levelwithin from about 5 minutes to about 15 minutes, from about 15 minutesto about 30 minutes, from about 30 minutes to 1 hour, from about 1 hourto about 2 hours, from about 2 hours to about 4 hours, from about 4hours to about 6 hours, or from about 6 hours to about 8 hours, or more,following administration of the ALDH agonist.

As another example, a subject ALDH agonist is administered to anindividual exhibiting drug toxicity, e.g., a toxic level of an aldehydefollowing ingestion, absorption, or inhalation of a drug (e.g., apharmaceutical compound, an illicit drug, etc.). In some embodiments,the aldehyde is produced following ingestion, absorption, or inhalationof a drug, by metabolism of the drug in the body. The toxic level ofaldehyde is reduced by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or at least about 90%, or more,compared to the level of the aldehyde in the individual before treatmentwith the ALDH agonist. In some embodiments, a subject ALDH agonist isadministered in an amount that is effective to reduce a toxic aldehydelevel by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90%, or more, within fromabout 5 minutes to about 15 minutes, from about 15 minutes to about 30minutes, from about 30 minutes to 1 hour, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, or from about 6 hours to about 8 hours, or more, followingadministration of the ALDH agonist. In some embodiments, a subject ALDHagonist is administered in an amount that is effective to reduce a toxicaldehyde level to a non-toxic level within from about 5 minutes to about15 minutes, from about 15 minutes to about 30 minutes, from about 30minutes to 1 hour, from about 1 hour to about 2 hours, from about 2hours to about 4 hours, from about 4 hours to about 6 hours, or fromabout 6 hours to about 8 hours, or more, following administration of theALDH agonist.

Methods of Reducing Salsolinol Levels

The present invention provides methods of reducing salsolinol levels inan individual, the methods generally involving administering to theindividual an effective amount of a subject ALDH agonist. Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquionoline) is acondensation product of dopamine with acetaldehyde. Acetaldehyde is ametabolic product of ethanol. Plasma salsolinol levels are higher inalcoholic compared to non-alcoholics. Reduction of salsolinol levels isuseful in reducing alcohol addiction.

In some embodiments, an effective amount of a subject ALDH agonist isadministered to an individual in need thereof following excessivealcohol (e.g., ethanol) consumption; where the effective amount providesfor a reduction in the levels of salsolinol in the individual of atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, or more, compared to thesalsolinol levels in the individual before treatment with the ALDHagonist. In some embodiments, an effective amount of a subject ALDHagonist is administered to an individual in need thereof at any time(e.g., not necessarily following excessive alcohol consumption). In someembodiments, a subject ALDH agonist is administered in an amount that iseffective to reduce a salsolinol level by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or at leastabout 90%, or more, within from about 5 minutes to about 15 minutes,from about 15 minutes to about 30 minutes, from about 30 minutes to 1hour, from about 1 hour to about 2 hours, from about 2 hours to about 4hours, from about 4 hours to about 6 hours, or from about 6 hours toabout 8 hours, or more, following administration of the ALDH agonist. Insome of these embodiments, the individual is one who has been diagnosedwith alcoholism. Symptoms and diagnosis of alcoholism are described in,e.g., Enoch and Goldman (2002) American Family Physician 65:441.

Methods of Treating Diabetes

The present invention provides methods of treating diabetes, the methodsgenerally involving administering to an individual in need thereof aneffective amount of a subject ALDH agonist. In some embodiments, asubject method of treating diabetes provides for treatment of a disorderthat is a result of diabetes, e.g., diabetic nephropathy, diabeticneuropathy, and the like.

In some embodiments, a subject ALDH agonist is administered in an amountthat is effective to reduce a blood glucose level in an individual,e.g., to reduce a blood glucose level in an individual by at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, or at least about 50% when comparedto the blood glucose levels in the absence of treatment with theagonist. In some embodiments, an effective amount of an ALDH agonist isan amount that is effective to reduce blood glucose levels to a normalrange. Normal fasting blood glucose levels are typically in the range offrom about 70 mg/dL to about 110 mg/dL before a meal. Normal bloodglucose levels 2 hours after a meal are usually less than about 120mg/dL. Normal blood glucose levels during an oral glucose tolerance test(involving drinking a sugar solution containing about 75 g glucose; thenmeasuring blood glucose levels at various times following drinking thesugar solution) include: less than 140 mg/dL 2 hours after drinking thesugar solution; and all readings between 0 and 2 hours after drinkingthe sugar solution less than 200 mg/dL. Blood glucose levels are alsosometimes expressed in mmol/L. Normal blood glucose levels are generallybetween about 4 mmol/L and 8 mmol/L. Normal blood glucose levels aregenerally less than about 10 mmol/L 90 minutes after a meal; and fromabout 4 mmol/L to about 7 mmol/L before meals.

In some embodiments, a subject treatment method comprises administeringa subject ALDH agonist, and co-administering at least a secondtherapeutic agent (e.g., insulin) for the treatment of diabetes. Insulinthat is suitable for use herein includes, but is not limited to, regularinsulin, semilente, NPH, lente, protamine zinc insulin (PZI),ultralente, insuline glargine, insulin aspart, acylated insulin,monomeric insulin, superactive insulin, hepatoselective insulin, and anyother insulin analog or derivative, and mixtures of any of theforegoing. Insulin that is suitable for use herein includes, but is notlimited to, the insulin forms disclosed in U.S. Pat. Nos. 4,992,417;4,992,418; 5,474,978; 5,514,646; 5,504,188; 5,547,929; 5,650,486;5,693,609; 5,700,662; 5,747,642; 5,922,675; 5,952,297; and 6,034,054;and published PCT applications WO 00/121197; WO 09/010645; and WO90/12814. Insulin analogs include, but are not limited to, superactiveinsulin analogs, monomeric insulins, and hepatospecific insulin analogs.

Methods of Treating Osteoporosis

The present invention provides methods of treating osteoporosis, themethods generally involving administering to an individual in needthereof an effective amount of a subject ALDH agonist. In someembodiments, an “effective amount” of an ALDH agonist is an amounteffective to increase bone density in the individual. In otherembodiments, an “effective amount” of an ALDH agonist is an amount thatis effective to reduce the rate of bone density loss.

Subjects Suitable for Treatment

Subjects suitable for treatment with a subject ALDH agonist includeindividuals suffering from a condition described above; individuals atrisk for developing a condition described above; individuals who havebeen treated for a condition described above with an agent other than asubject ALDH agonist, and who either failed to respond to suchtreatment, or who initially responded to such treatment, butsubsequently relapsed; individuals who are refractory to treatment withan agent other than a subject ALDH agonist for a condition describedabove; and individuals who cannot tolerate treatment with an agent otherthan a subject ALDH agonist for a condition described above. Subjectssuitable for treatment with a subject compound include individuals whohave been diagnosed with a condition as discussed above.

Methods Involving Administering an ALDH Agonist

A subject treatment method involving administration of a subject ALDHagonist is suitable for treating various conditions, as noted above,including disorders or conditions associated with or resulting fromoxidative stress; disorders or conditions associated with nitroglycerininsensitivity; disorders or conditions associated with toxic levels ofethyl alcohol, aldehyde, methanol, ethylene glycol monomethyl ether,biogenic or xenogenic aldehydes, etc.; and heart diseases andconditions, such as coronary artery disease, angina, etc. In someembodiments, the individual is a human who is homozygous for an ALDH2allele that encodes an ALDH2 having an amino acid sequence as depictedin FIG. 1A. In other embodiments, the individual is a human who carriesone or two ALDH2*2 alleles, where an ALDH2*2 allele encodes an ALDH2having the E487K variant as depicted in FIG. 1B.

Approximately 40% of the East Asian population carries the semidominantALDH2*2 allele. Such individuals can be characterized by a response toethanol consumption that includes one or more of facial flushing,nausea, and tachycardia. In addition, ALDH2*2 individuals are also lessresponsive to nitroglycerin treatment for such disorders as angina andcoronary artery disease. Individuals who are heterozygous or homozygousfor the ALDH2*2 allele are suitable for treatment with a subject methodinvolving administration of a subject ALDH agonist.

Methods of Treating Conditions Associated with Ischemic Stress

Subjects suitable for treatment with subject ALDH agonist includeindividuals who are scheduled to undergo cardiac surgery or who haveundergone cardiac surgery; individuals who have experienced a stroke;individuals who have suffered brain trauma; individuals who haveprolonged surgery; individuals who have suffered a myocardial infarct(e.g., acute myocardial infarction); individuals who suffer fromcerebrovascular disease; individuals who have spinal cord injury;individuals having a subarachnoid hemorrhage; and individuals who willbe subjected to organ transplantation. Subjects suitable for treatmentwith a subject ALDH agonist also include individuals having an ischemiclimb disorder, e.g., resulting from Type 1 or Type 2 diabetes.

Methods of Treating Acute Free-Radical Associated Diseases

Subjects suitable for treatment with subject ALDH agonist includeindividuals who are having or who have experienced a seizure;individuals having skin damage resulting from UV exposure; individualshaving photodamage of the skin; individuals having an acute thermal skinburn injury; and individuals suffering from tissue hyperoxia.

Methods of Treating Chronic Free-Radical Associated Diseases

Subjects suitable for treatment with subject ALDH agonist includeindividuals who have been diagnosed with Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, or otherneurodegenerative disease; individuals having atherosclerosis;individuals having esophageal cancer; individuals having head and necksquamous cell carcinoma; and individuals having upper aerodigestivetract cancer.

Methods of Treating Cardiac Conditions

Subjects suitable for treatment with a subject ALDH agonist includeindividuals having angina; individuals having heart failure; individualswho exhibit an insensitivity to nitroglycerin in the treatment of anginaor heart failure; individuals having hypertension; and individualshaving heart disease.

Detoxification Methods

Subjects suitable for treatment with a subject ALDH agonist includeindividuals who have toxic levels of an aldehyde, e.g., via ingestion ofa toxic compound, via inhalation of a toxic compound, via ingestion orinhalation of toxic levels of a compound, or via production of thealdehyde during normal metabolism. Such individuals include, but are notlimited to, individuals who have ingested or inhaled ethanol, methanol,ethylene glycol monomethyl ether, or other xenogenic or biogenicaldehyde compounds. For example, such individuals include individualswho have ingested or inhaled pesticides, fungicides, or other suchcompounds; individuals who have consumed excessive levels of ethanol;and the like.

Methods of Treating Diabetes

Subjects suitable for treatment with a subject ALDH agonist includeindividuals having Type 1 or Type 2 diabetes. Subjects suitable fortreatment include individuals who have been diagnosed with Type 1diabetes mellitus, where such individuals include those having a fastingblood glucose level greater than about 126 mg/dL. Such individualsinclude those having blood glucose levels of greater than about 200mg/dL following a two-hour glucose tolerance test (75 g anhydrousglucose orally). Subjects suitable for treatment include individuals whohave been diagnosed with Type 2 diabetes; individuals who have not yetbeen diagnosed with Type 2 diabetes, but who are at risk of developingType 2 diabetes, e.g., individuals having a body mass index (weight inkilograms divided by height (in meters) squared) greater than 25, e.g.,individuals having a body mass index from about 25 to about 27, fromabout 27 to about 30, or greater than 30.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating an ALDH-mediated disorderselected from alcohol intolerance, alcohol addiction, alcohol abusedisorder, methanol poisoning, and ethylene glycol monomethyl etherpoisoning, the method comprising administering to individual in needthereof a compound Formula I:

wherein: each of R₁, R₂, and R₃ is independently selected from H, ahalo, a substituted or unsubstituted phenyl group, an aliphatic group,an alkyl group, a substituted alkyl group, an alkenyl group, an alkynylgroup, a substituted or unsubstituted cyclic group, a substituted orunsubstituted heterocyclic group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted heteroaryl group; A is C or S,wherein a=1 when A=C, and a=2 when A=S; Ar₁ is selected from asubstituted heterocyclic group, an unsubstituted heterocyclic group, asubstituted aryl group, an unsubstituted aryl group, a substitutedheteroaryl group, and an unsubstituted heteroaryl group; and Ar₂ is asubstituted pyridine oxide; or a pharmaceutically acceptable salt, ananalog, or a derivative thereof.
 2. The method of claim 1, wherein Ar₁is:

wherein R₄-R₈ are each independently selected from H, a halo, asubstituted or unsubstituted phenyl group, an aliphatic group, an alkylgroup, a substituted alkyl group, an alkenyl group, an alkynyl group, asubstituted or unsubstituted cyclic group, a substituted orunsubstituted heterocyclic group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted heteroaryl group.
 3. Themethod of claim 1, wherein Ar₁ is a substituted or unsubstitutedheterocyclic group.
 4. The method claim 1, wherein the substitutedpyridine oxide is selected from:

and R₉ to R₁₂ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup.
 5. The method of claim 1, wherein the compound is of Formula Ia:

wherein: X_(n) and X_(y) are each independently H, C, N, O, or ahalogen; . . . (dotted line) is an optional bond; z is the integer 0, 1,or 2, with the provisos that: 1) z=0 when X=halogen and . . . is not abond; and 2) when z=0, X=O, is not a bond, and one or more oxygen atoms(X) are present, oxygen is attached to a methyl group; n is the integer0 or 1; y is the integer 0 or 1; A=C or S, wherein a=1 when A=C, and a=2when A=S; Ar is a substituted pyridine oxide of a formula selected from:

 and R₉ to R₁₂ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup; or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 6. The method of claim 1, wherein the compound is of FormulaII:

wherein: X_(n) and X_(y) are each independently H, C, N, O, or ahalogen; n is the integer 0 or 1; y is the integer 0 or 1; . . . (dottedline) is an optional bond; z is the integer 0, 1, or 2; A is C or S,wherein a=1 when A=C, and a=2 when A=S; Ar is a substituted pyridineoxide; and R₁ to R₆ are each independently selected from H, a halo, asubstituted or unsubstituted phenyl group, an aliphatic group, an alkylgroup, a substituted alkyl group, an alkenyl group, an alkynyl group, asubstituted or unsubstituted cyclic group, a substituted orunsubstituted heterocyclic group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted heteroaryl group; or apharmaceutically acceptable salt, an analog, or a derivative thereof. 7.The method of claim 6, wherein the substituted pyridine oxide is of aformula selected from:

and R₉ to R₁₂ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup.
 8. The method of claim 1, wherein the compound is of Formula a:

wherein R₁ to R₄ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup; or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 9. The method of claim 8, wherein the compound has a structureselected from:

or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 10. The method of claim 1, wherein the compound is of Formulab:

wherein R₁ to R₄ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup; or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 11. The method of claim 10, wherein the compound has astructure selected from:

or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 12. The method of claim 1, wherein the compound is of Formulac:

wherein R₁ to R₄ are each independently selected from H, a halo, analiphatic group, an alkyl group, a substituted alkyl group, an alkenylgroup, an alkynyl group, a substituted or unsubstituted cyclic group, anether, a substituted or unsubstituted amine, an ester, and an amidegroup; or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 13. The method of claim 12, wherein the compound has astructure selected from:

or a pharmaceutically acceptable salt, an analog, or a derivativethereof.
 14. The method of claim 1, wherein the compound is present in apharmaceutical composition comprising the compound and apharmaceutically acceptable excipient.
 15. The method of claim 1,wherein the compound is administered by a route selected fromintramuscular, intravenous, subcutaneous, and oral.
 16. The method ofclaim 1, wherein the individual has an ALDH2*2 allele.
 17. The method ofclaim 14, wherein the pharmaceutical composition administered by a routeselected from intramuscular, intravenous, subcutaneous, and oral. 18.The method of claim 14, wherein the individual has an ALDH2*2 allele.19. The method of claim 3, wherein Ar₁ is selected from a substituted orunsubstituted pyridine, a thiazole, an imidazole, a thiophene, aquinoline, an isoquinoline, and a furan group.